ResultsA carocin S2-defective mutant, TF1-2, was obtained by Tn5 insertional mutagenesis using F-rif-18. A 5706-bp DNA fragment was detected by Southern blotting, selected from a genomic DNA library, and cloned to the vector, pMS2KI. Two adjacent complete open reading frames within pMS2KI were sequenced, characterized, and identified as caroS2K and caroS2I, which respectively encode the killing protein and immunity protein. Notably, carocin S2 could be expressed not only in the mutant TF1-2 but also in Escherichia coli DH5α after entry of the plasmid pMS2KI. Furthermore, the C-terminal domain of CaroS2K was homologous to the nuclease domains of colicin D and klebicin D.

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Moreover, SDS-PAGE analysis showed that the relative mass of CaroS2K was 85 kDa and that of CaroS2I was 10 kDa. The phytopathogenic enterobacterium, Pectobacterium carotovorum subsp. Carotovorum, is a phytoparasitic, Gram-negative, facultative anaerobic bacterium. Pcc produces many extracellular pectic enzymes (pectate lyase, pectin lyase, exopolygalacturnoate lyase) and hydrolytic enzymes causing soft-rot disease, tissue maceration, and cell wall collapse ,.

The only current strategy against soft-rot disease involves chemical agents that unavoidably contaminate the environment. Kikumoto et al. Have demonstrated that mixed bacteriocin-producing avirulent strains of Pcc show high efficacy against soft-rot disease of Chinese cabbage.Bacteriocins are bactericidal, extracellular toxins, produced by both Gram-positive and Gram-negative bacteria ,. These proteinaceous molecules kill closely related bacteria. The susceptible cell is recognized by specific target receptors on the membrane, and the producer cell evades lethality by expressing a cognate immune protein.

The colicin family produced by Escherichia coli is divided into DNase (colicins E2, E7, E8 and E9), RNase (colicins E3, E4 and E6), tRNase (colicins D and E5), and pore-forming colicins (colicins A, E1, Ia and Ib). Bacteriocins (especially nuclease bacteriocins) have a high amino acid sequence homology.Natural bacteriocin molecules act via a number of mechanisms. For example, colicin E3 is a well-known ribonuclease that specifically cleaves 16S rRNA at the 3'-end of the coding sequence both in vivo and in vitro, which leads to the abolishment of protein synthesis resulting in death of the susceptible cell ,. Previous reports indicate that colicin E3 consists of a killer protein with three domains (i.e., a translocation domain T domain, receptor binding domain R domain, and nuclease domain) and an immunity protein that retards antibiotic activity ,. The R domain recognizes a specific receptor, BtuB on the cell membrane and the T domain interacts with the TolB protein in the cell periplasm of the sensitive cell to facilitate entry of the killer domain through the cell membrane. In addition to the attack mechanism, the immunity mechanism has been extensively elucidated. Notably the immunity protein and the killer protein interact initially at very high affinity because of charge attraction, and are separated at the cell surface through energy generated from the proton motif force ,.In general, the C-terminal domain determines the type of bacteriocin.

The C-terminal nuclease domains are not only interchangeable but also lack species specificity. Strikingly, the tRNase type of bacteriocin may accelerate exhaustion of tRNA in the cytoplasmic pool and thereby impair protein synthesis in vivo. Have demonstrated that particular tRNA molecules can be digested by colicin D as well as by colicin E5 ,. It has been suggested that phage-associated klebicin D is a tRNase type of bacteriocin based on similarity to the nuclease-like domain of colicin D.Nguyen et al. Reported production of a high-molecular-weight bacteriocin (carotovoricin Er) and Chuang et al. Reported production of a low-molecular-weight bacteriocin (LMWB; carocin) by Pectobacterium,. The former has a bulky antenna-like tail, inner core, and contractile cylindrical structure, and the carotovoricin-caused inhibition zone can be easily distinguished from that of carocin by its low diffusibility.

Carocin S1 is a deoxyribonuclease type of LMWB (indicated by the letter S) and is secreted by Pcc strain 89-H-4. Additionally, export of Carocin S1 utilizes the type III secretion system in Pcc, which also controls the cell motility of the bacterium.Pcc strain F-rif-18 is a spontaneous rifampin-resistant mutant of the wild-type 3F-3. Ultraviolet radiation can induce Pcc strain F-rif-18 to produce the LMWB Carocin S2. One of several sensitive cells, SP33, was selected as an indicator strain here. In the present study, the chromosomal bacteriocin gene, carocin S2, was introduced into an expression plasmid encoding two proteins, CaroS2K and CaroS2I.

These proteins were purified and characterized and their primary activities of killing (CaroS2K) and immunity (CaroS2I) were investigated in vivo and in vitro. Conjugation between F-rif-18 and E. Coli 1830 resulted in 3,500 colonies after selection on Modified Drigalski's agar medium containing rifampin and kanamycin. In bacteriocin assay, the size of the inhibition zone around each isolate was compared with that of F-rif-18.

Mutant colonies were identified by smaller inhibition zones. This evidence of mutation suggested that transposon Tn 5 had been inserted into LMW bacteriocin-related genes. The strain TF1-2, a putative insertion mutant, would no longer produce LMW bacteriocin (Figure ).

Figure 1Bacteriocin assays of Tn 5 insertion mutants of Pcc strains. Strain number: 1, 3F3 (wild type); 2, 1830 ( E. Coli); 3, F-rif-18 (parent); 4, TF1-1 and 5, TF1-2 (insertion mutant). Other unlabelled strains are Tn 5 insertion mutants of F-rif-18 strain. The indicator is Pcc strain SP33.To ascertain whether Tn 5 was actually introduced into the genomic DNA of putative isolates, the nptII gene of isolates was amplified using two primers P3 and P4. Southern blot technology showed that Tn 5 had been inserted (Additional file, Figure S1). Identification of Tn5-inserted DNA Structures.

To identify Tn 5-interrupted genes, genomic DNA from TF1-2 was amplified with TAIL-PCR using an array of specific primers (Additional file, Figure S8). A 2621-bp DNA fragment, including two open reading frames (ORFs), was identified as the sequence containing the bacteriocin structural gene. This gene was designated the carocin S2 gene. To characterize the carocin S2 gene, the TF1-2 probe was designed to hybridize in Southern blots with a Bam HI-digested DNA fragment from the genomic library of F-rif-18 (Figure ). A 5706-bp Bam HI-digested DNA fragment (Figure ), harboring two complete ORFs of carocin S2, was cloned into the plasmid pMCL210 (Additional file, Figure S2). The carocin-producing plasmid was designated as pMS2KI.

The amplicon, comprising the predicted ORF2 of caroS2I, was subcloned into the pGEM-T easy vector, resulting in the plasmid pGS2I (Additional file, Figure S5). Figure 2DNA library screening and scheme of carocin S2 gene.

(A) The TF1-2 probe was used to screen DNA fragments from the genomic DNA library of F-rif-18. The DNA was digested with various restriction enzymes as follows: 1. HindIII; 3 HpaI; 4. DNA leader marker; C. The TF1-2 probe DNA.

The arrowhead indicates the 5.7-kb carocin S2 fragment. (B) Shown is the 5.7-kb segment of DNA containing the carocin S2. The location of TF1-2 probe and part amplicon of cDNA of caroS2K and caroS2I were shown. Transcriptional analysis and in vivo expression of carocin S2 gene. Figure 3Reverse Transcription PCR of RNA.

Shown are cDNA from the following strains: Lanes 1, F-rif-18; 2, TF1-2; 3, TF1-2/pMS2KI, 4, DH5α; 5, DH5α/pMS2KI.; 6, SP33; 7, SP33/pGS2I. The amplicons of caroS2K and caroS2I are 925 bp and 259 bp, respectively. The corresponding amplicons of 16S rRNA from the examined strains (lower panel). All samples were loaded equally.The presence of the 925-bp amplicon revealed that caroS2K was being transcribed in the cell (panel caroS2K in Figure ).

The TF1-2 strain, which is a Tn 5 insertional mutant, could not transcribe caroS2K (lane 2), but the ability of TF1-2 to transcribe caroS2K was restored by introduction of pMS2KI (lane 3). It was apparent that the amount of caroS2K expression was dependent on the number of copies of plasmid pMS2KI (compare lane 1 to lane 3). Additionally, carocin S2 can be expressed in E. Coli strain DH5α by introduction of pMS2KI (lane 4 and lane 5). The presence of a 259-bp amplicon showed that caroS2I was transcribed constitutively (panel caroS2I in Figure ). The caroS2I gene was transcribed unexpectedly in mutant strain TF1-2 even though the plasmid pMS2KI was introduced (lane 3). This demonstrated that caroS2I is expressed constitutively regardless of whether the gene caros2K is transcribed.

Possibly an individual promoter for caroS2I gene is located behind the Tn 5 insertion site in the caroS2K gene. CaroS2I transcripts were detected in strain SP33 with plasmid pGS2I (lanes 6 and 7). Although both the SP33 strains (with or without pGEM T-easy) were susceptible to Carocin S2, SP33/pGS2I appeared to grow in the presence of CaroS2K (Figure ). Figure 4Recovery and immunity activity of carocin S2.

(A) Antibacterial activity of carocin S2 from different strains. The indicator was Pcc strain SP33.

Strain number: 1, F-rif-18; 2, TF1-2; 3, TF1-2/pMS2KI; 4, DH5α/pMS2KI; 5, DH5α. (B) Assay for caroS2I. The colony and inoculated strains were F-rif-18.

The indicator strains were: 1, SP33; 2, SP33/pGEM-T easy; 3, SP33/pGS2I.To prove that pMS2KI contained the gene for Carocin S2, pMS2KI was introduced into TF1-2 and E. Both TF1-2/pMS2KI and DH5α/pMS2KI had ability to express the activity of Carocin S2 (Figure ).

The size of inhibition zone around strain TF1-2/pMS2KI was equal to that around DH5α/pMS2KI but still smaller than that around the wild-type strain F-rif-18. On the other hand, the quantity of transcripts expressed in vivo and in vitrodid not usually correspond. Deduction of the amino acid sequence of Carocin S2The carocin S2 gene consists of two ORFs (Additional file, Figure S7): one containing the 2352-bp caroS2K gene and the other containing the 273-bp caroS2I gene. The stop codon (TGA) of caroS2K overlaps the first start codon of caroS2I by 4-bp (ATGA).

The amino sequences were deduced from the nucleotide sequence of the carocin S2 gene using DNASIS-Mac software (HITACHI, Japan) and compared to other analogous proteins using the BLAST and FASTA search tools. ORF1 was found to encode a 783-amino acid protein with a high degree of homology to Pcc21 carocin D, Escherichia coli colicin D and Klebsiella oxytoca klebicin D (Figure ); ORF2 was found to encode a 90-amino acid protein that shows homology to the immunity proteins of colicin D and klebicin D (Figure ). Thus, caroS2K produces an antibiotic with a deduced molecular mass of 85 kDa. CaroS2I (a 10-kDa protein of 90 amino acids) was shown to confer resistance to CaroS2K. It is particularly noteworthy that the homology between CaroS2K and Colicin D and Klebicin D is at the C-terminal end of these proteins where the catalytic center of a ribonuclease is located. According to the FASTA program, the amino acid segment between Asp677 and the C-terminus of CaroS2K shares almost 60% similarity with the minimal tRNase domain of colicin D and klebicin D (Figure ). Since the colicin D and klebicin D are well-known tRNase family of bacteriocins, suggests that Carocin S2 might therefore be a ribonuclease.

Figure 5Region similarity of the putative domains of carocin S2 with those of related bacteriocins. The related ORFs are shown. Percentage values indicate the percent relatedness to the corresponding regions in carocin S2. The length of each domain is proportional to the number of amino acids. Homologous domains are shaded similarly. Domain I is homologous with the N-terminal T domain of colicin E3. Domain II resembles the receptor binding domains of other bacteriocins, but has no significant homology to other sequences in the database ,.

Domain III and ORF2 of carocin S2 are highly homologous to colicin D and klebicin D. Purification and characterization of Carocin S2.

Coli BL21 (DE3) recombinants, which were transformed with pES2KI or pES2I, were used to express CaroS2K protein or CaroS2I protein individually. Coomassie blue stained SDS-PAGE gels of purified Carocin S2 are shown in Figure. The band corresponding to CaroS2K was purified. The gel indicates a relative mass (M r) of about 85 kDa (Figure ), enrichment of the purified CaroS2K (arrowhead), and disappearance of other bands. Purification of CaroS2I by the same procedure resulted in a more intense band in the region of M r 10 kDa (arrowhead; Figure ). Figure 6SDS-PAGE analysis of purified protein.

Shown are the CaroS2K (A) and CaroS2I (B). Samples were subjected to electrophoresis in 10% polyacrylamide gels, which were stained with Coomassie blue. Lane M, molecular weight standards (kDa); lane 1, cell lysate of E. Coli BL21/pET32a; lane 4, cell lysate of BL21/pET30b; lanes 2 and 5, IPTG-induced cell lysates of BL21/pES2kI and BL21/pES2I, respectively; lanes 3 and 6, purified protein obtained after elution. The arrowheads indicate the killing protein of carocin S2K (A) and the immunity protein of carocin S2I (B).

Figure 8In vitro hydrolysis of DNA and RNA by Carocin S2. (A) Analysis of the DNase activity of carocin S2. Lane M, the HindIII-digested λ DNA marker; lane 1, genomic DNA only; lanes 2 and 3, genomic DNA treated or untreated with carocin S2 in buffer, respectively; lane 4, equal quantity of EcoRI-digested genomic DNA. The 5'-labeled total RNA (B) and 3'-labeled total RNA (C) (1 μg of RNA per sample) were incubated without (lane 1) or with 1 μg (lane 2), 100 ng (lane 3), 10 ng (lane 4), or 1 ng (lane 5) of Carocin S2 and the result was assessed by autoradiography.

The arrowhead indicates that the RNA segment digested from ribosome. Equal amounts of Carocin S2I and Carocin S2K mixed before RNA digestion (lane 6).Surprisingly the RNA segments were larger when the RNA was 3'- 32P-labeled compared with 5'- 32P-labeling (Figures and ). As the concentrations of 23S RNA and 16S RNA decrease on the addition of increasing concentrations of CaroS2K, it is assumed that more ribosomal RNA is degraded leaving material that is ostensibly the ribosome. When excess concentrations of caroS2K (i.e 1 μg) are added then most of the ribosomal RNA is degraded leading to a destabilization and subsequent degradation of the ribosome (Figure, lane 2).

We hence consider that CaroS2K (in sufficient amount) would degrade the ribosome. CaroS2I inhibits the killing activity of CaroS2K because a mixture of equal quantities of CaroS2K and CaroS2I prevented digestion of RNA segments by CaroS2K (Figure, lane 6).Subsequently, treatment of the genomic DNA of the indicator strain SP33 with the purified CaroS2K protein had no effect on deoxyribonuclease activity, as compared to the pattern of EcoRI-digested genomic DNA (Figure and Additional file, Figure S4). Nucleotide sequence accession numberThe Genbank accession number of the sequence of the carocin S2 gene is HM475143. In this study, a chromosome-borne gene encoding bacteriocin, carocin S2, in Pcc strain 3F3 was shown to possess ribonuclease activity. According to Bradley's classification, Carocin S2 is a low-molecular-weight bacteriocin.

Two genes, caroS2K and caroS2I, encode the 85-kDa and 10-kDa components, respectively, of Carocin S2. The substrate and gene structure of carocin S2 were unlike those of other bacteriocins from Pcc.On the basis of sequence analysis, carocin S2 comprises these two overlapping ORFs, caroS2K and caroS2I (Additional file, Figure S7). A putative Shine-Dalgarno sequence 5'-AUGGA-3', which has also been seen in the DNA sequence of carocin S1, is located upstream (-9 bp to -13 bp) of the start codon AUG, suggesting that it could be a ribosome binding site for caroS2K. Comparison of the upstream sequences of both caroS2K and caroS2I has shown that the two consensus sequences, 5'-TATAAAAA-3' (-34 bp to -41 bp) and 5'-GAAGT-3' (-61 bp to -65 bp), are both upstream from the start codon. Presumably, 5'-TATAAAAA-3' is the -10 promoter and 5'-GAAGT-3' is the -35 promoter for the carocin S2 gene, even though they differ from those of E. Coli.A putative -10 promoter is 33 bp upstream from the initiator ATG of the caroS2K gene, in which the SD sequence is embedded, while the -35 promoter is 19 bp upstream of the -10 promoter region. The putative promoter of the -35 box of caroS2I is located similarly near the -10 box, but the -10 box is just 24 bp upstream of the start codon where no SD sequence is apparent.

Although those hypothesized promoters are located within the caroS2K structural gene, transcripts of caroS2I are routinely produced (Figure ). This suggests that caroS2I RNA expression may be regulated posttranscriptionally, in spite of close neighboring genes downstream of the gene caroS2K; that is, core promoter elements may influence the expression of caroS2I gene.In the present study, we attempted to separate CaroS2K from CaroS2I attached to (His) 6-tag using a Nickel column (pEH2KI; Additional file, Figure S5), but a small amount of CaroS2I (Mr 10 kDa) was observed in SDS-PAGE gels (Figure, bottom in lane 3), which had little influence on the activity of CaroS2K as the purified protein still had transient killing activity. Additionally, the activity of the Carocin S2 complex at 4℃ was long-lasting indicating good stability.The C-terminal amino acid sequence of Carocin S2 had higher homology to those of colicin D and klebicin D, which are produced by E. Coli and Klebsiella oxytoca, respectively, than to the amino acid sequence of carocin S1 from the same species (Additional file, Figure S6B).The amino acid sequence of CaroS2K has three putative domains. Domain I (the N-terminal 314-residue sequence ending in Pro314) is regarded as the translocation domain and is homologous to the translocation domains of carocin D and colicin E3 (Figure ). It is assumed to direct the cytotoxic domain to the periplasmic space ,.

Additionally, the putative TonB box (a sequence recognition motif DTMTV) was found in the N-terminal domain of CarocinS2, which is thought to participate in bacteriocin translocation. Thus, we suggested that Carocin S2 could be a TonB-dependent bacteriocin.Domain III (extending from Asp677 to the carboxyl terminus) is the killer domain. Particularly noteworthy is the resemblance of the killer domain to the tRNase domain of colicin D and klebicin D (Figure ), and thus we suggested that carocin S2 might have tRNase activity ,.

Domain II extends 141 residues from Ilu315 to Val455 and is hypothesized to be the binding site that recognizes specific receptors on cell membranes. Additionally, domain III has no significant homology to carocin D, suggesting that carocin S2 and carocin D have different functions.Finally, we showed that total RNA (whether labeled with radioactive phosphate at the 5'- or at the 3'- end) is sensitive to Carocin S2.

Carocin S2 degraded 5'-labeled total RNA but not 5'-labeled CaroS2K-free RNA (Figure ), and the amount of degradation was not dose-dependent (arrowhead). However, the appearance of segments of unknown origin paralleled partial degradation of 23S and 16S rRNA (Figure ). These results suggest that the site of excision (either conformational or sequential) is close to the 5'-terminus of rRNA. Notably, the decrease in the amount of rRNA depended on the amount of Carocin S2 protein present, with complete degradation occurring in the presence of excess Carocin S2. Reported that RNase type of bacteriocins, colicin E3 and colicin E5, catalyze the hydrolysis of the shorter RNAs from 16S rRNA ,.

Moreover, colicin E5 was found to hydrolyze tRNA in vitro. Furthermore, it was previously reported that colicin E3 cleaved 16S rRNA completely, and even 30S rRNA ,. In our study, carocin S2 acted as an RNase that hydrolyzes rRNA (both 23S and 16S) in vitro. In terms of enzymatic function, Carocin S2 may act as an endo- and exo-ribonuclease simultaneously.

Moreover, CaroS2I significantly inhibited nuclease activity in vitro but not in vivo (Figures, Figure andAdditional file, Figure S3). We speculated that immunity protein CaroS2I might not be able to cross the cell membrane, as previously described. Although our in vitro experiment showed that carocin S2 was a ribonuclease, further investigation is needed to clarify its function in cells.One of the other Tn 5 insertional mutants, TF1-1, which disrupted the coding sequence of the fliC gene, was found to halt expression of Carocin S2 (Figure ), indicating that Carocin S2 can also be secreted via the type III secretion system. The role of carocin S2 as an RNase in the cytoplasm is to prevent protein synthesis by cleaving either 23S rRNA or 16S rRNA.

The role of the immunity protein, CaroS2I, is usually to stop the damage caused by CaroS2K in the cytoplasm. More details of the actual mechanism of carocin S2 remain to be elucidated. Strain or plasmidDescriptionSourceEscherichia coli1830pro¯ met¯ Kan r Nm r, containing transposon Tn5 on the suicidal plasmid pBJ4JI DH5αsupE44ΔlacU169(Φ80lacZΔM15) hsdR17recA1 gyrA96thi-1relA1 BL21(DE3)hsdS gal(λ cI ts857 ind1 Sam 7 nin5 l ac UV5-T7 gene 1) Pectobacterium carotovorum subsp. Kan r: Kanamycin; Cml r: Chloramphenicol; Rif r: Rifampicin; Amp r: Ampicillin.: See Additional file, Figure S5. Bacterial conjugationOvernight cultures of Pcc (recipient) and E. Coli (donor) were mixed and spread onto 0.22-μm membrane filters placed on LB agar media and incubated overnight at 28°C. The progeny after conjugation were appropriately diluted and cultivated on Modified Drigalski's medium (with ampicillin and kanamycin 100 μg ml -1) overnight at 28°C.

All isolates were placed on IFO-802 medium and tested for bacteriocins. Bacteriocin was assayed using the double-layer method, and Pcc SP33 was used as indicator strain. The cells were incubated for 12 hours to form colonies, exposed to ultraviolet irradiation, incubated again for 12 hours, treated with chloroform to kill the cells, and then covered with soft agar containing indicator cells. The bacteriocin production was indicated by a zone of inhibition of indicator-cell (SP33) growth around the colony. Genetic-engineering techniqueThe procedures of plasmid preparation, genomic DNA isolation, and DNA manipulation were performed as described by Sambrook et al. Oligonucleotide DNA primers were synthesized by MD Bio Inc. (Taipei, Taiwan).

The PCR was amplified with Go-Taq DNA polymerase (Promega, USA). The thermal asymmetric interlaced PCR (TAIL-PCR) was performed as previously described.Plasmids were introduced into Pcc strains using electroporation (1.25 kV/cm, 200 Ω, 25 μF).

For heat-shock transformation, the competent cells of E. Coli were prepared according to the method of Hanahan.Exponentially growing cells (OD 595 of about 6.0) were harvested for RNA preparation. Total RNA was isolated using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA was resuspended in diethylpyrocarbonate (DEPC)-treated water. The concentration of RNA was determined by OD 260 absorption, and RNA was analyzed by electrophoresis on 1.5% formaldehyde-morpholinepropanesulfonic-agarose gel.Reverse transcription-PCR (RT-PCR) was carried out with AMV Reverse Transcriptase (Promega Inc., Taiwan) according to manufacturer's instructions.

RNA (1 μg) was subjected to RT-PCR containing CaroS2re1 used as a reverse primer in first-strand cDNA synthesis. The RT mixtures were diluted and used as templates in a PCR reaction with two primers CaroS2re1 and CaroS2for1 (Additional file, Table S1).A 2621-bp BamHI- HindIII digested DNA fragment, including the caroS2K and caroS2I genes, was amplified from pMS2KI with primers of CarocinS2Kfor2 and CarocinS2Irev2 (Additional file, Table S1) and subcloned into pET32a to give the plasmid pEN2K (Additional file, Figure S5). The pES2KI was obtained by excision of the Tag element between the rbs (ribosome binding site) and start code (for CaroS2K) in pEN2K using the SLIM method as previously described ,.

The 5IHT32a2KIforT, 5IHTGT2KIforS, 5IHT32a3KIrevT, and 5IHT32a4KIrevS primers were used. A 273-bp fragment of the caroS2I gene was amplified by PCR and ligated into the NdeI and XhoI site of pET30b to form the plasmid pEC2I.

Similarly, the plasmid pES2I was obtained by deleting the (His) 6-tag of pEC2I (carried out as described above with primers of X21forT, X21forS, X21revT and X21revS). Subsequently, pES2KI and pES2I were introduced into E. Coli BL21 (DE3) cells, respectively. Restriction DNA library screening and Southern blotsSouthern blots were performed according to the DIG Application Manual (Roche, USA). A 543-bp DNA fragment (TF1-2 probe) was amplified with TF1-2P and TF1-2A2 primers (Additional file, Table S1), subcloned into pGEM-T Easy vector (Promega Inc., USA), and labeled using a Random Primed DNA Labeling Kit (Roche Diagnostics, USA).The genomic DNA of the wild-type strain F-rif-18 was digested with various restriction endonucleases, with sites located outside the putative open reading frame. Samples were electrophoresed and analyzed with Southern blotting. After detection using the TF1-2 probe, the DNA from positive gel slices was purified and cloned into pMCL210 to give the carocin-producing plasmid pMS2KI.

The pMS2KI construct was isolated and detected as above with the TF1-2 probe. Protein purificationThe transformant cells of BL21, harboring pES2KI or pES2I, were grown in 500 ml to an OD 595 of 0.4.

The cells were induced with isopropyl-β-D-thiogalactopyranoside (IPTG; final concentration, 0.1 mM; at 25°C for 12 h). Subsequently, the cells were pelleted and the pellets were sonicated (10 cycles of 9 s with 9-s intervals). BL21/pES2KI pellets were subjected to ammonium sulfate precipitation (30-40%), resuspended in buffer A (30 mM NaCl and 20 mM Tris-Cl, pH 8.0), and applied to a Fractogel column (Merck, USA). The fraction was eluted by a NaCl gradient (30 mM-1.4 M). After purification through a P-100 size-exclusion column (BioRad, USA), the CaroS2K fractions were pooled and concentrated using an Amicon centriprep-50 column (Millipore, USA) and dissolved in buffer A.

BL21/pES2I pellets were precipitated by ammonium sulfate (70-100%) and resuspended in buffer A. CaroS2I purification involved a similar chromatographic procedure using the Amicon centriprep-3 column (Millipore, USA). The concentration of protein was determined by the Bradford assay (Amresco, USA). In vitro determination of Carocin S2 activityTotal RNA was treated with calf intestinal alkaline phosphatase (Promega, USA) at 55°C for 30 min as recommended by the manufacturer. The reaction was arrested by adding 5 mM nitrilotriacetic acid, and RNA was extracted with equal volumes of phenol/chloroform.

An aliquot of phosphatase-treated RNA was 5'- 32P-labeled at 37°C for 30 min by incubation with a mixture of γ- 32PATP, T4 polynucleotide kinase (Promega Inc, USA), and reaction buffer in nuclease-free water. 5'- 32PCytidine 3',5'-bisphosphate (pCp) and T4 RNA ligase (Promega, USA) were used for 3'-labeling of RNA.

Subsequently, the mixture was purified by MicroSpin G-25 columns (GE Healthcare, USA). The purified labeled RNA was divided into aliquots and incubated without or with Carocin S2 at 28°C for 60 min, respectively. To measure its activity, CaroS2I was pre-mixed with an equal amount of CaroS2K. The mixtures were subjected to electrophoresis on a 9% polyacrylamide gel (19:1) containing 7M urea, 50 mM Tris, 50 mM boric acid, and 1 mM EDTA, pH 8.3. All samples were electrophoresed at 15℃ by PROTEIN II xi (BioRad, USA).To confirm DNase activity, 1 μg of genomic DNA from SP33 in solution containing buffer A was incubated with or without Carocin S2 at 28°C for 90 min. An equal quantity of genomic DNA was digested with EcoRI at 28°C for 90 min. Samples were then subjected to electrophoresis on 1% agarose gel.

Antibiotic activity of Carocin S2Overnight cultures of SP33 were diluted (1:100) with LB medium and grown at 28°C to a density of approximately 10 5 CFU ml -1. The activity of increasing concentrations of Carocin S2 on cells in suspension incubated at 28°C for 60 min was assessed. CaroS2I was pre-mixed with an equal molar ratio of CaroS2K. All reaction mixtures were spread onto LB agar plates and incubated at 28°C for 16 h. The experiment was performed three times.

Colonies growing on a series of plates were respectively counted. Computer analysis of sequence dataSequencing of the DNA fragments was carried out using an ABI automated DNA sequencer 373S. The nucleotide sequence data were compiled by DNASIS-Mac software (Hitachi, Japan). Amino acid sequences were compared using international BLAST and FASTA servers. Also, the putative domains of Carocin S2 were predicted using the PSI/PHI-BLAST. AcknowledgementsThe support of this work by grants from the National Science Council (grants NSC-97-2313-B-005-027-MY3) of Taiwan (R.O.C.) is gratefully acknowledged.

Authors' contributionsYC participated in the discovery and characterization of Carocin S2, and he wrote this manuscript. JL participated in protein purification. HP participated in manuscript preparation. KC supported the Pcc strain SP33 and for insightful discussion and guidance. DY conceived of the study, participated in its design, and corrected the manuscript. All authors read and approved the final version of the manuscript.

Supplementary material. Additional file 1: Figure S1. Analysis of Tn5 insertional mutants by southern blotting. Lane M, the HindIII-digested λ DNA marker; the genomic DNA of strains were loading as follows: lane 1, TF1-2; lane 2, F-rif-18; lane 3, 3F3; lane 4, TF1-1. Lane 5, the construct pGnptII that contain the detect probe DNA nptII. The result shows that TF1-2 and TF1-1 was a Tn 5 insertional mutant. The construct pMS2KI was cloned from genomic DNA library and screening by southern blotting with TF1-2 probe.

By southern blotting, it showed that the carocin S2 has been cloned to form pMS2KI. The total RNA of SP33 were digested with Carocin S2 and electrophoresis as follows: lane 1, RNA (1 μg); lane 2, RNA and CaroS2K (20 μg); lane 3, RNA and CaroS2I (4 μg); lanes 4 to 6 are RNA (1 μg) and CaroS2K (20 μg) with gradient concentration of CaroS2I, which were added with 4 μg (lane 4); 20 μg (lane 5); 100 μg (lane 6). All reactions were performed at 28℃ for 3 hours. Metal effect of In vitro hydrolysis of DNA by Carocin S2.

Lane M, the HindIII-digested λ DNA marker; lane 1, the genomic DNA of SP33 only; lane 2, the EcoRI-digested genomic DNA; the genomic DNA was incubated with Carocin S2 (lane 3 to 5), or not. Magnesium acetate, nickel acetate and zinc acetate was added in buffer A (pH = 7), respectively.

The reactions were performed at performed at 28℃ for 1 hour. Schematic representation of the cloning strategy used in this study. (1) A 543-bp amplicon was cloned into the vector pTF1 to form the pTF1-2-probe. (2) The TF1-2 probe was prepared. (3) The multi-enzyme-digested DNA fragments were obtained from F-rif-18 genomic DNA, and they were detected on southern blots. (4) Positive cDNA was cloned into the carocin-producing plasmid pMS2KI. (5) A 2621-bp amplicon, from pMS2KI, was subcloned into pET32a to form pEN2K.

(6) The 5'-transcriptional element, which would be translated into the Flag tag, was deleted from pEN2K using the SLIM method. (7) By using SLIM method, an element encoding a stretch of six histidines was inserted into caroS2I to form pEH2KI. (8) A 484-bp amplicon was subcloned into pGEM T-easy vector to form pGS2I. (9) A273-bp fragment of the caroS2I gene was amplified from pGS2I and subcloned into pET30b to form pECS2I. (10) The 3'-transcriptional element, which would be translated to (His) 6-Flag, was deleted from pES2I using the SLIM method. Alignment of the deduced amino acid sequences of carocin S2 with those of homologous domains of bacteriocins. The potential TonB-binding motif is shown by red underline.

(A) The N-terminal translocation domain of CaroS2K (Met1 to Pro314) has homology to carocin D and colicin E3. (B) The killing domain of CaroS2K (Asp677 to carboxyl terminus) has homology to the minimal tRNase domain of colicin D and klebicin D. (C) The deduced amino acid of immunity protein of CaroS2I has homology to colicin D and klebicin D. The gene and deduced amino acid sequence of carocin S2 shows in the study. The sequence was truncated form pMS2KI. The underline shows the putative promoter.

Schematic representation of thermal asymmetric interlaced PCR (TAIL-PCR) were manipulated according to the method of Liu and Whittier, but the annealing temperature was decreased from 63℃ to 60℃ for specific primers ,. Amplifying the unknown DNA fragment are the specific primers which are complementary to the known sequence (Tn5) and the arbitrary degenerate primers which could be complementary to the opposite unknown site. The specific primers (SP) are PR1, PR2, PR3, PF1, PF2, PF3, and TF1-2S1 to TF1-2A6 primers for opposite direction (Additional file, Table S1).

In addition, the arbitrary degenerate primers (AD) N1, N2, and N3 were respectively used as simultaneous PCR amplification (see above).

The amino acid motifs available to enzymic catalysts are diverse. Still, many enzymes fall into classes wherein great similarities exist. One well-studied class is that of the serine proteases. This class of enzymes has an active-site motif known as the catalytic triad, which is composed of the residues Ser⋯His⋯Asp linked by hydrogen bonds (, ).The importance ascribed to the Ser⋯His⋯Asp motif is in large part due to its presence in the active sites of nonhomologous serine proteases.

In recent years, the triad has also been found in several lipases (, ), Fusarium solani cutinase , and the Sindbis virus core protein. Replacing the aspartate residue in the catalytic triad of human lipoprotein lipase with a glycine is responsible for familial Type I hyperlipoproteinemia.Similar to the catalytic triad, a His⋯Asp motif is found in the active sites of serine carboxypeptidase , acetylcholinesterase , phospholipase A 2 , haloalkane dehalogenase , dienelactone hydrolase , a variety of zinc-dependent enzymes , and pancreatic ribonucleases (, ). This His⋯Asp motif is known as the “catalytic dyad”.

With the exception of the pancreatic ribonucleases, enzymes containing the His⋯Asp dyad have syn-oriented carboxylates , though the benefit to catalysis of a syn vs anti orientation appears to be minimal (, ).In all enzymes containing a catalytic triad or dyad, an hydroxyl group functions as a nucleophile. The role of the histidine residue is to abstract a proton from the hydroxyl group and thereby to increase the nucleophilicity of the oxygen. The role of the aspartate residue has been the subject of much study and debate, as has the importance of the catalytic triad in general.The catalytic triad was once thought to act in a “charge relay mechanism”. Specifically, it was postulated that a proton was removed from the serine residue by histidine while another proton was being removed from histidine by aspartate (, ). The unperturbed p K a of aspartic acid is too low to favor such a mechanism, and the general consensus today is that the charge relay mechanism is not operative. Instead, the aspartate residue forms a hydrogen bond that could serve both to orient the histidine sidechain and to increase its p K a, enhancing its ability to abstract a proton from the serine residue.Site-directed mutagenesis has been used to illuminate the role and importance of the aspartate residue in the catalytic triad of two serine proteases.

Replacing the aspartate residue with asparagine in trypsin (, ) or alanine in subtilisin leads to a 10 4-fold reduction in catalytic activity. This decrease suggests that the aspartate residue plays a critical role in catalysis. X-ray diffraction analysis of the trypsin variant revealed, however, that the catalytic histidine residue was being stabilized as an unproductive tautomer by its interaction with asparagine. This result suggests that one role of the aspartate residue in a Ser⋯His⋯Asp triad is to orient the proper tautomer of histidine.Two attempts have thus far been made to determine the role of Asp121, the aspartate in the catalytic dyad of bovine pancreatic ribonuclease A RNase A; E.C.

3.1.27.5;. In one study, Asp121 was replaced with asparagine in a semisynthetic enzyme. This semisynthetic RNase A, RNase(1 – 118).(111 – 124), consists of a noncovalent complex between residues 1 – 118 of RNase A (obtained from proteolytic digestion of RNase A), and an overlapping synthetic peptide composed of the fourteen C-terminal residues of RNase A, except with Asp121 replaced by an asparagine residue.

The semisynthetic D121N analog has 5% of the catalytic activity of the analogous wild-type semisynthetic enzyme. A difficulty with interpreting the results of this study, however, is that the three-dimensional structure of the semisynthetic D121N analog exhibits numerous changes compared to that of the analogous wild-type semisynthetic enzyme (, ). In another study, site-directed mutagenesis was used to replace Asp121 in RNase A itself with a glutamate residue. The D121E enzyme has 17% of the activity of the wild-type enzyme for the hydrolysis of 0.1 mM Cp and 16% for the hydrolysis of 0.4 mM Cp.

No other characterization of this variant has been reported. In related work, replacing Asp116 of the His⋯Asp dyad of angiogenin, a homolog of RNase A, with an asparagine or alanine residue was shown to increase ribonucleolytic activity by 8- and 15-fold, respectively.Here, we address the role of the aspartate residue in the catalytic dyad of RNase A. RNase A catalyzes the two-step hydrolysis of the P–O 5 ′ bond of RNA on the 3′ side of pyrimidine residues. Depicts the classical mechanism for these reactions. In the transphosphorylation step, His119 of the His⋯Asp dyad acts as an acid. The slow hydrolysis of the 2′,3′-cyclic phosphate occurs separately and resembles the reverse of transphosphorylation (, ). It is in this second step that the action of the catalytic dyad of RNase A most resembles that of the catalytic triad.

The amino acid sequences of pancreatic ribonucleases from over forty vertebrates are known (, ), and are evolving rapidly. The catalytic dyad is conserved in each of these ribonucleases, suggesting that these residues are important. We have created RNase A variants in which Asp121 is replaced with an asparagine or alanine residue. Here, we report on the three-dimensional structures of the variants, as well as their abilities to catalyze transphosphorylation and hydrolysis. MaterialsEscherichia coli strain BL21(DE3) (F − ompT r B-m B −) was from Novagen (Madison, WI). Buffers (except Tris), cUMP, and IPTG were from Sigma Chemical (St. Tris was from Fisher Scientific (Pittsburgh, PA).

Poly(C) was from Midland Reagent (Midland, TX). UpA was synthesized by J. Thompson using methods published previously (, ).

Growth media were from Difco (Detroit, MI) or, for 10-L growths, Marcor Development (Hackensack, NJ). DNA sequences were determined with the Sequenase Version 2.0 kit from U.S.

Biochemicals (Cleveland, OH). DEAE Sephadex A-25 anion exchange resin, S-Sepharose cation exchange resin, and the mono-S FPLC cation exchange column were from Pharmacia LKB (Piscataway, NJ).

Bacterial terrific broth TB; contained (in 1 L) tryptone (12 g), yeast extract (24 g), glycerol (4 mL), KH 2PO 4 (2.3 g), and K 2HPO 4 (12.5 g). MutagenesisPreviously, we described the construction of a bovine pancreatic cDNA library, the cloning of the cDNA that codes for RNase A, and the efficient expression of this cDNA in Escherichia coli. Here, the codon for Asp121 was changed to one for an asparagine or alanine residue by oligonucleotide-mediated site-directed mutagenesis using the oligonucleotides: 5′–ACTACTACAC GCTAGC GTTAAAGTGGACT–3′ and 5′–ACTACTACAC GCTAGC GGCAAAGTGGACT–3′, where the underlined bases differ from those in the wild-type cDNA. The GCT mismatch introduced a unique, translationally silent NheI site that was used to screen for mutated plasmids.

The complete sequence of the cDNA that codes for each enzyme was determined. Plasmids that direct the expression of wild-type, D121N, and D121A RNase A were transformed into E.

Coli BL21(DE3). Production of RNase A in E. ColiTB (0.20 L) containing ampicillin (200 μg/mL) was inoculated with a culture frozen in aqueous glycerol (30% w/v). After growth overnight at 37 °C, cells were collected by centrifugation and suspended in 0.20 L of fresh TB. This suspension was used to inoculate 10 L of TB containing ampicillin (200 μg/mL). When A = 4 at 600 nm, IPTG was added to a final concentration of 0.5 mM.

The culture was grown for an additional 3.5 h.Cells were collected by centrifugation, resuspended in 0.10 L of cold TE buffer, and lysed by passage through a French pressure cell twice. The lysate was centrifuged at 30000 g for 30 min. The resulting pellet was washed with a cold solution (0.30 L) of deoxycholate (1 mg/mL), and centrifuged again. The pellet was suspended in 0.50 L of Tris-acetic acid buffer, pH 8.0, containing urea (9 M), acetic acid (50 mM), sarcosine (40 mM), and EDTA (1 mM). Sonication was used to aid dissolution. To reduce any disulfide bonds, dithiothreitol was added to a final concentration of 50 mM. After 1 h, the solution was passed through DEAE A-25 resin (25 g) that had been equilibrated with the above resuspension solution, and acetic acid (one tenth volume) was added to the effluent.

The resulting solution was dialyzed exhaustively against 20 mM acetic acid. The dialysate was centrifuged, the supernatant was diluted to 1 L, and the pH raised to 8.1 by the addition of Tris base. Reduced glutatione and oxidized glutathione were then added to final concentrations of 2.3 mM and 0.8 mM, respectively.

After 24 h the resulting solution was loaded on to an S-Sepharose cation exchange column (0.20 L). The column was washed with 1 L of 50 mM HEPES buffer, pH 7.9, and the adsorbed proteins were eluted with a linear gradient (0.50 L + 0.50 L) of NaCl (0 – 0.50 M).

Fractions were pooled on the basis of A at 280 nm, and the pooled fractions were diluted with buffer to a protein concentration of. Enzyme KineticsSteady-state kinetic parameters for the cleavage of UpA and poly(C), and the hydrolysis of cUMP were determined by spectrophotometric assays. The catalytic dyad of RNase A most resembles the well-characterized catalytic triad of serine proteases during the hydrolysis reaction. For this reason, the steady-state kinetic parameters for the hydrolysis reaction were evaluated as a function of pH.Assays were performed at 25 °C in a 0.10 M solution of the appropriate buffer, adjusted in pH with aqueous NaOH or HCl. Enough NaCl was added to each reaction to increase the ionic strength to I = 0.10 M, which was calculated based on the concentrations of buffer components and substrate. The buffer used for assays of the transphosphorylation of UpA (0.10 – 1.0 mM) and poly(C) (0.050 – 1.0 mM) was MES (pH 6.0). The buffers used for assays of the hydrolysis of cUMP were formic acid (pH 3.45, 4.00), acetic acid (4.48, 5.02), MES (5.53, 6.03), Bis-Tris (6.00, 7.02), BICINE (6.48, 7.97, 8.46), MOPS (6.94, 7.46), and AMPSO (9.00).Assays were performed in a 1.0- or 0.2-cm cuvette with a Cary Model 3 spectrophotometer equipped with a Cary temperature controller.

Substrate concentrations were determined by ultraviolet absorption using ε 260 = 24,600 M −1cm −1 at pH 7.0 for UpA and ε 268 = 6200 M −1cm −1 at pH 7.8 for poly(C) , or by mass for cUMP. The concentrations of substrate in the assays ranged from K m/5 to 5 × K m. BSA (0.5 mg/mL) was added to the reactions containing poly(C). The cleavage of UpA was monitored at 286 nm using Δε = −755 M −1cm −1. The cleavage of poly(C) was monitored at 238 nm.

The Δε at 250 nm, as calculated from the difference in molar absorptivity of the polymeric substrate and the mononucleotide cyclic phosphate product, has been reported to be 2380 M −1cm −1 at pH 6.2. The Δε at 238 nm was determined to be 2792 M −1cm −1 by observing the change in absorption of a partially cleaved substrate at 250 nm and 238 nm. The hydrolysis of cUMP was monitored at either 282 nm or 293 nm.

The values for the extinction coefficients determined for the hydrolysis of cUMP are listed in. Kinetic Data AnalysisData processing and nonlinear regression analysis were performed with the program MATHCAD (MathSoft; Cambridge, MA). Initial velocity data were weighted in proportion to the magnitude of each data point. The pH–rate profiles were fitted by using the logarithm of the kinetic constants. Reported errors were generated by the asymptotic variance – covariance matrix method.

Because of strong product inhibition and a relatively small change in extinction coefficient, the initial velocities for the hydrolysis of cUMP were difficult to measure reliably. For this reason the steady-state kinetic parameters for this substrate were determined by using the integrated rate equation (, ) to fit the complete time-course of each hydrolysis reaction. The use of the integrated rate equation provided more accurate values for the steady-state kinetic parameters as well as values for K p, the dissociation constant for the RNase A.3′-UMP complex. Each progress curve for the hydrolysis reaction was fitted to the integrated rate equation with the inclusion of product inhibition by an iterative procedure in which the kinetic parameters were varied by hand and the fit judged by eye.

The value of k cat was allowed to vary slightly from progress curve to progress curve to allow for small errors in enzyme concentration, with the values of K m and K p being held constant. The derivative of the functions so obtained was used to determine better fitting values for the initial velocities. These velocities were then used to obtain the kinetic parameters using the Michaelis-Menten equation. If the parameters so realized differed greatly from the initial values, the process was repeated until they differed little from the prior round. Generally, one or two rounds were sufficient. Structural differences between D121N RNase A and D121A RNase A and their semisynthetic analogs.

(A) Stereoview of the overlap of the active sites of D121N RNase A (thick red lines) and the semisynthetic D121N analog thin blue lines;. The sD121N analog contains a sulfate ion in its active site. (B) Stereoview of the overlap of the active sites of D121A RNase A (thick red lines) and the sD121A analog thin blue lines;. D121A RNase A contains an acetate ion in its active site, and the sD121A analog contains a sulfate ion in its active site. Water molecules are depicted for the variants (red closed spheres) and semisynthetic analogs (blue open spheres). This figure was created with the program MOLSCRIPT.

SubstrateRNase Ak cat (s −1)%K m (mM)%k cat/ K m(mM −1s −1)%UpAWild-Type890 ± 301000.38 ± 0.031002300 ± 190100D121N190 ± 20211.6 ± 0.2420120 ± 205D121A30 ± 33.42.2 ± 0.358014 ± 20.6poly(C)Wild-Type475 ± 101000.14 ± 0.011003300 ± 200100D121N51 ± 2110.10 ± 0.0171510 ± 6015D121A8.2 ± 0.31.70.20 ± 0.0214040 ± 41.2cUMPWild-Type3.7 ± 0.21002.3 ± 0.51001.6 ± 0.3100D121N1.55 ± 0.11422.7 ± 0.71200.58 ± 0.1036D121A0.187 ± 0.0065.11.6 ± 0.2700.11 ± 0.017. (3)where H+ is the hydrogen ion concentration; k cat/ K m int, k cat int, k cat int2, and K p int are pH-independent constants; K a, K b, K c, and K d are the acid dissociation constants of uncomplexed enzyme; K p is the acid dissociation constant of uncomplexed 3′-UMP; and those constants with an “s” or “p” subscript are for the enzyme.substrate or enzyme.product complex (, ).

In, the terms associated with K c and K d were not included in the fit when there was no indication of their presence. As no indication for p K p,p was present, it was set at 2.0. The value of p K p was set at 5.8. The free enzyme values of K p were obtained from the k cat/ K m profiles. The p K a values and intrinsic parameters calculated from eq – are listed in. AValues were calculated by fitting the data in to eq –.As shown in, wild-type RNase A, D121N RNase A, and D121A RNase A have virtually identical k cat/ K m profiles except for an alkaline dip in the profile of the D121N enzyme. Both wild-type RNase A and D121N RNase A display a second low pH limb at a pH = 4.As shown in, wild-type RNase A, D121N RNase A, and D121A RNase A have similar k cat profiles at pH 7, the k cat profile of the wild-type enzyme reaches a plateau, but those of the variants decrease.

The k cat profiles for wild-type RNase A and D121N RNase A have slope = 1 at pH. Plot of log( k cat) vs pH for the hydrolysis of cUMP by wild-type RNase A (E), D121N RNase A (I), and D121A RNase A (D). The curves are nonlinear least-squares fits of the data to. To simplify comparisons, the values of k cat for D121N RNase A and D121A RNase A were multiplied by scaling factors of 1.6 and 16, respectively.As shown in, the K p profile of D121N RNase A is similar to that of the wild-type enzyme except for a decreased K p at high pH. The K p of D121A RNase A is twofold less than that of the other two enzymes. Three-Dimensional StructuresRNase A was the third enzyme (after lysozyme and carboxypeptidase) whose structure was solved by X-ray diffraction analysis. Despite this early precedent , the crystalline structures reported here are the first for active-site variants.

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These structures show that replacing residue Asp121 of the His⋯Asp catalytic dyad with an asparagine or alanine residue does not affect the structure of RNase A beyond residue 121. Interpretations of function based on the identity of the active-site residues alone are thus justified.The structure of RNase A is comprised of a twisted four-stranded, antiparallel β-sheet consisting of two long central β-strands, β2 and β3 (residues 71 – 92 and 94 – 110, respectively), flanked by two shorter β-strands, β1 and β4 (residues 41 – 48 and 118 – 123, respectively).

Three helicies are found in RNase A: α1 at the N-terminus (residues 3 – 13), and α2 and α3 nearly perpendicular to and at either end of the β-sheet (residues 24 – 34 and 50 – 60, respectively). Nucleotide binding occurs in a deep cleft created by α1 and β3. The bases of adjacent RNA nucleotides bind in three enzymic subsites referred to as B1, B2, and B3. Catalysis occurs in the P1 subsite and results in the cleavage of the P–O 5′ bond specifically on the 3′-side of a pyrimidine nucleotide bound in the B1 subsite. Residues from the B1 subsite and P1 catalytic site compose the walls and floor of the groove. Gln11, Lys41, and Asn44 form one wall, and His119, Asp121, and Ala122 form the other.

His12, Asn44, Thr45, and Phe120 form the floor of the B1 subsite. This discussion will focus on structural deviations observed in residues constituting the active site (Figures and ). Catalysis by D121N RNase A and D121A RNase AThe effect of replacing Asp121 on k cat/ K m and k cat is small, but significant (Tables – ). Substitution with an asparagine residue decreases the rate constants for transphosphorylation by 10 1-fold and those for hydrolysis by 10 0.5-fold.

Substitution with an alanine residue decreases the rate constants for transphosphorylation by 10 2-fold and those for hydrolysis by 10 1-fold (Tables – ). The differential effects on transphosphorylation and hydrolysis are still greater than these values suggest because a chemical step does not limit the rate of cleavage of UpA or poly(C). We conclude that the contribution of Asp121 to transphosphorylation is greater than that to hydrolysis.The small contribution of Asp121 to catalysis belies the importance of the hydrogen bond between His119 and Asp121. This contribution is similar to that observed in an analogous study on the structure and function of phosphatidylinositol-specific phospholipase C. Yet, a similar His⋯Asp dyad has been proposed to form a low-barrier hydrogen bond of extraordinary strength during catalysis by the protease chymotrypsin (, ), though this interpretation is controversial.

One criterion for such a bond is an 1H chemical shift of 17 – 20 ppm. The 1H chemical shift of N ε2H of His119 appears at a much higher field. PH-Dependence of CatalysisThe overall shape of the pH–rate profiles for catalysis by RNase A can be interpreted by using a model in which the only important protonation states are those of the two active-site histidine residues, which act in concert during catalysis ;. Accordingly, log–log plots of the pH–rate profiles for k cat/ K m and k cat are expected to be bell-shaped with slopes approaching unity. The k cat/ K m profiles that have been reported are indeed bell-shaped, but the shape of the k cat profiles varies with the substrate (-).

In general, workers who have used cCMP as the substrate have found that the slope = 1 for the acidic leg of the k cat profiles; but those who have used cUMP have found that the slope 7 than are reported by others (, ).Values of p K a for the two histidine residues of wild-type RNase A can be derived from the k cat/ K m profile. The values agree well with results from NMR spectroscopy (-, ).

Differences for any given p K a value are within error, especially when recalling that when two p K a’s have similar values, the average value is much better determined from pH–rate profiles than are the individual values. The p K a values of the D121N and D121A enzymes that are derived from pH–rate profiles do not deviate significantly from those of wild-type RNase A, at least at I = 0.10 M. Role of Asp121 in CatalysispH–rate profiles for catalysis by D121N RNase A are similar to those for catalysis by the wild-type enzyme (Figures – ). This similarity suggests that the same titratable groups are participating in catalysis and that these groups have similar p K a values. Yet in the structure of D121N RNase A, N δ2 rather than O δ1 of Asn121 is oriented toward His119. This orientation appears to stabilize a neutral form of the imidazolyl sidechain in which N ε2 is unprotonated and N δ1 is protonated.

This tautomer of His119 is likely to be ineffective, either as an acid during the transphosphorylation reaction or as a base during the hydrolysis reaction. The major role of Asp121 thus appears to be to orient the proper tautomer of His119 for catalysis.

This role is similar to that assigned to the aspartate residue in the catalytic triad of trypsin. In the D121A enzyme, Ala121 cannot interact with the imidazolyl group of His119.

Yet, D121N RNase A is a better catalyst than is D121A RNase A (Tables – ). Thus, the His119⋯Asn121 interaction observed in the crystalline D121N enzyme (Figures and ) is unlikely to exist in solution. Rather, Asn121 is likely to move such that His119 can effect catalysis.

ConclusionsReplacing Asp121, a residue that is conserved in pancreatic ribonucleases, with an asparagine or alanine residue has no effect on the overall three-dimensional structure of RNase A. Replacing Asp121 decreases the rate of transphosphorylation by ≤10 2-fold and the rate of hydrolysis by ≤10 1-fold. Thus, Asp121 of RNase A makes a significant but not a substantial contribution to catalysis. The likely role of Asp121 is simply to position the proper tautomer of His119, which Asn121 in the crystalline D121N enzyme fails to do.

We speculate that the role of Asp121 in catalysis alone is unlikely to warrant its complete conservation in pancreatic ribonucleases.