Mutational analysis of tyrosine-191 in the catalysis of Cephalosporium acremonium isopenicillin N synthase

Isopenicillin N synthase (IPNS) is a key enzyme responsible for the catalytic conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N in the beta-lactam antibiotic biosynthetic pathway. The Aspergillus nidulans IPNS crystal structure implicated amino acid residues tyrosine-189, arginine-279, and serine-281 in the substrate-binding of the valine carboxylate portion of ACV via hydrogen bonds. In previous reports, we provided mutational evidence for the critical involvement of the corresponding arginine-281 and serine-283, which constitute a conserved R-X-S motif, for the catalysis of Cephalosporium acremonium IPNS (cIPNS). In this study, we report the site-directed mutagenesis of the corresponding tyrosine-191 in cIPNS to four amino acids from different amino acid groups, namely, phenylalanine, serine, histidine, and aspartate. The mutants Y191F, Y191H, and Y191R respectively yielded specific activities at levels of 3, 8.6, and 18.8% relative to the wild-type when enzyme bioassays were performed using purified protein fractions. These results were surprising, as previous mutational analyses involving arginine-281 and serine-283 resulted in non-measurable specific activities, thus suggesting that tyrosine-191 is important but not critical for the activity of cIPNS due to its involvement in ACV binding. Hence, it is likely that tyrosine-191 is the least critical of the three residues involved in binding the ACV valine carboxylate moiety.     

Glutamine-230 influences enzyme solubility but not catalysis in Streptomyces clavuligerus isopenicillin N synthase

The conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N is dependent upon the catalytic action of isopenicillin N synthase (IPNS), an important enzyme in the penicillin and cephalosporin biosynthetic pathway. Recent catalytic investigations on the conserved glutamine-230 in the bacterial Streptomyces jumonjinensis IPNS and the corresponding glutamine-234 in the fungal Cephalosporium acremonium IPNS showed contrasting results whereby the former was suggested to be essential for IPNS activity whereas the latter was found not to be so. In order to unravel these conflicting results, we report the site-directed mutagenesis investigation on the corresponding glutamine-230 in a third IPNS isozyme, which is the bacterial Streptomyces clavuligerus IPNS (scIPNS). IPNS enzymatic assays showed that catalytic activity of the mutant Q230L scIPNS was reduced but not eliminated. Moreover, the solubility of the mutant enzyme was also markedly reduced. Hence, we can conclude that glutamine-230 in scIPNS is not essential for catalysis and correspondingly in all IPNS.     

Histidine-272 of isopenicillin N synthase of Cephalosporium acremonium, which is possibly involved in iron binding, is essential for its catalytic activity

Abstract Amino acid sequence alignment of the Cephalosporium acremonium isopenicillin N synthase (cIPNS) to similar non-heme Fe²⁺-containing enzymes from 28 different sources (bacterial, fungal, plant and animals) revealed a homologous region of high sequence conservation containing an invariant histidine residue at position 272 in cIPNS. The importance of this histidine residue in cIPNS was investigated through site-directed mutagenesis by replacing the histidine residue with leucine. The mutated gene was verified by DNA sequence analysis and expressed in Escherichia coli . When analyzed by denaturing gel electrophoresis and immunoblotting, the mutant cIPNS had identical mobility as that of the wild-type enzyme. Enzyme studies on the mutant enzyme showed loss of enzymatic activity indicating that His272 is essential for the catalytic function of cIPNS, possibly as a ligand for iron binding.     

Mutational analysis of conserved glycines 42 and 256 in Cephalosporium acremonium isopenicillin N synthase.

Isopenicillin N synthase (IPNS) is critical for the catalytic conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine to isopenicillin N in the penicillin and cephalosporin biosynthetic pathway. Two conserved glycine residues in Cephalosporium acremonium IPNS (cIPNS), namely glycine-42 and glycine-256, were identified by multiple sequence alignment and investigated by site-directed mutagenesis to study the effect of the substitution on catalysis. Our study showed that both the mutations from glycine to alanine or to serine reduced the catalytic activity of cIPNS and affected its soluble expression in a heterologous host at 37 degrees C. Soluble expression was restored at a reduced temperature of 25 degrees C, and thus, it is possible that these glycine residues may have a role in maintaining the local protein structure and are critical for the soluble expression of cIPNS.     

An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation

Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB’s catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by fine-tuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism.     

Site-directed mutagenesis of arginine-89 supports the role of its guanidino side-chain in substrate binding by Cephalosporium acremonium isopenicillin N synthase

Isopenicillin N synthase (IPNS) catalyses a key step in the penicillin and cephalosporin biosynthetic pathway which involves the oxidative cyclisation of the acyclic peptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N. Based on crystallographic evidence from the Aspergillus nidulans IPNS crystal structure complexed with the substrate ACV (Roach et al. (1997) Nature 387, 827-830), we were able to provide mutational evidence for the critical involvement of the conserved R-X-S motif in ACV binding in IPNS. The crystal structure further implicated arginine-87 in the binding of the aminoadipyl portion of ACV. Thus, in this study, the site-directed mutagenesis of the corresponding arginine-89 in Cephalosporium acremonium IPNS (cIPNS) was performed to ascertain its role in cIPNS. Alteration of arginine-89 to five amino acids from different amino acid groups, namely lysine, serine, alanine, aspartate and leucine, was performed and no activity was detected in all the mutants obtained when enzyme bioassays were performed. Furthermore, the solubility of the mutants was considerably lower than the wild-type cIPNS after expression at 37 degrees C, but could be recovered when the expression temperature was lowered to 25 degrees C. This suggests that arginine-89 could be critical for the activity of cIPNS due to its involvement in ACV binding and the solubility of wild-type enzyme.     

Mutation of N304 to leucine in Streptomyces clavuligerus deacetoxycephalosporin C synthase creates an enzyme with increased penicillin analogue conversion

Superimposition of deacetoxycephalosporin C synthase (DAOCS) and isopenicillin N synthase (IPNS) structures revealed that R74, R160, R266 and N304 are strategically located in the catalytic cavity of Streptomyces clavuligerus DAOCS (scDAOCS) and are crucial for orchestrating different substrates. Substitutions at these sites to a hydrophobic leucine residue were expected to stabilize the hydrophobic substrate bound state. Substantial improvements in the biotransformation of penicillin G, ampicillin and amoxicillin to their respective cephalosporin moieties were observed using the N304L mutant scDAOCS. Thus, our results have demonstrated the enhancement of scDAOCS activity via critical computational analysis and site-directed mutagenesis of endogenous ligands.     

Arg‐85 and Thr‐430 in murine 5‐aminolevulinate synthase coordinate acyl‐CoA‐binding and contribute to substrate specificity

5‐Aminolevulinate synthase (ALAS) controls the rate‐limiting step of heme biosynthesis in mammals by catalyzing the condensation of succinyl‐coenzyme A and glycine to produce 5‐aminolevulinate, coenzyme‐A (CoA), and carbon dioxide. ALAS is a member of the α‐oxoamine synthase family of pyridoxal 5′‐phosphate (PLP)‐dependent enzymes and shares high degree of structural similarity and reaction mechanism with the other members of the family. The X‐ray crystal structure of ALAS from Rhodobacter capsulatus reveals that the alkanoate component of succinyl‐CoA is coordinated by a conserved arginine and a threonine. The functions of the corresponding acyl‐CoA‐binding residues in murine erthyroid ALAS (R85 and T430) in relation to acyl‐CoA binding and substrate discrimination were examined using site‐directed mutagenesis and a series of CoA‐derivatives. The catalytic efficiency of the R85L variant with octanoyl‐CoA was 66‐fold higher than that of the wild‐type protein, supporting the proposal of this residue as key in discriminating substrate binding. Substitution of the acyl‐CoA‐binding residues with hydrophobic amino acids caused a ligand‐induced negative dichroic band at 420 nm in the CD spectra, suggesting that these residues affect substrate‐mediated changes to the PLP microenvironment. Transient kinetic analyses of the R85K variant‐catalyzed reactions confirm that this substitution decreases microscopic rates associated with formation and decay of a key reaction intermediate and show that the nature of the acyl‐CoA tail seriously affect product binding. These results show that the bifurcate interaction of the carboxylate moiety of succinyl‐CoA with R85 and T430 is an important determinant in ALAS function and may play a role in substrate specificity.     

Amino acid residues in the GIY-YIG endonuclease II of phage T4 affecting sequence recognition and binding as well as catalysis

Phage T4 endonuclease II (EndoII), a GIY-YIG endonuclease lacking a carboxy-terminal DNA-binding domain, was subjected to site-directed mutagenesis to investigate roles of individual amino acids in substrate recognition, binding, and catalysis. The structure of EndoII was modeled on that of UvrC. We found catalytic roles for residues in the putative catalytic surface (G49, R57, E118, and N130) similar to those described for I-TevI and UvrC; in addition, these residues were found to be important for substrate recognition and binding. The conserved glycine (G49) and arginine (R57) were essential for normal sequence recognition. Our results are in agreement with a role for these residues in forming the DNA-binding surface and exposing the substrate scissile bond at the active site. The conserved asparagine (N130) and an adjacent proline (P127) likely contribute to positioning the catalytic domain correctly. Enzymes in the EndoII subfamily of GIY-YIG endonucleases share a strongly conserved middle region (MR, residues 72 to 93, likely helical and possibly substituting for heterologous helices in I-TevI and UvrC) and a less strongly conserved N-terminal region (residues 12 to 24). Most of the conserved residues in these two regions appeared to contribute to binding strength without affecting the mode of substrate binding at the catalytic surface. EndoII K76, part of a conserved NUMOD3 DNA-binding motif of homing endonucleases found to overlap the MR, affected both sequence recognition and catalysis, suggesting a more direct involvement in positioning the substrate. Our data thus suggest roles for the MR and residues conserved in GIY-YIG enzymes in recognizing and binding the substrate.     

Electron spin echo envelope modulation studies of the Cu(II)-substituted derivative of isopenicillin N synthase: a structural and spectroscopic model.

Electron spin echo envelope modulation spectroscopy (ESEEM) was used to study the active site structure of isopenicillin N synthase (IPNS) from Cephalosporium acremonium with Cu(II) as a spectroscopic probe. Fourier transform of the stimulated electron spin-echo envelope for the Cu(II)-substituted enzyme, Cu(II)IPNS, revealed two nearly magnetically equivalent, equatorially coordinated His imidazoles. The superhyperfine coupling constant, Aiso, for the remote 14N of each imidazole was 1.65 MHz. The binding of substrate to the enzyme altered the magnetic coupling so that Aiso is 1.30 MHz for one nitrogen and 2.16 MHz for the other. From a comparison of the ESEEM of Cu(II)IPNS in D2O and H2O, it is suggested that water is a ligand of Cu(II) and this is displaced upon the addition of substrate.     

Cloning, sequence analysis and transcriptional study of the isopenicillin N synthase of Penicillium chrysogenum AS-P-78

A gene (ips) encoding the isopenicillin N synthase of Penicillium chrysogenum AS-P-78 was cloned in a 3.9 kb SalI fragment using a probe corresponding to the amino-terminal end of the enzyme. The SalI fragment was trimmed down to a 1.3 kb NcoI-BglII fragment that contained an open reading frame of 996 nucleotides encoding a polypeptide of 331 amino acids with an Mr of 38012 dalton. The predicted polypeptide encoded by the ips gene of strain AS-P-78 contains a tyrosine at position 195, whereas the gene of the high penicillin producing strain 23X-80-269-37-2 shows an isoleucine at the same position. The ips gene is expressed in Escherichia coli minicells using the lambda phage PL promoter. Some similar sequence motifs were found in the upstream region of the ips gene of P. chrysogenum when compared with the upstream sequences of the ips genes of Cephalosporium acremonium and Aspergillus nidulans. Primer extension studies indicated that the start of the mRNA coincides with a T in position -11 which is located in a conserved pyrimidine-rich sequence, near two CAAG boxes. Clones of P. chrysogenum Wis 54-1255 transformed with the ips gene showed a five-fold higher isopenicillin N synthase activity than the untransformed cultures.     

Cell-free conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine into an antibiotic with the properties of isopenicillin N in Cephalosporium acremonium.

Cell-free extracts of antibiotic-negative mutants of Cephalosporium acremonium converted delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (LLD-tripeptide) into an antibiotic that was destroyed by penicillinase. The enzymic activity of the extracts was destroyed by boiling, but was not inhibited by cycloheximide. LLL-Tripeptide was totally inactive as substrate. The product resembled isopenicillin N, but not penicillin N, in its antibacterial spectrum. We propose that isopenicillin N is the first product of cyclization of LLD-tripeptide.     

Site-directed mutagenesis of mouse thymidylate synthase: alteration of Arg44 to Val44 in a conserved loop guarding the active site has striking effects on catalysis and nucleotide binding

The arginine located at position 44 of mouse thymidylate synthase is in a highly conserved loop that is in close proximity to the active site cleft of the enzyme. Structural analyses have suggested that this arginine forms hydrogen bonds with the alpha-carboxylate of the C-terminal amino acid and the phosphate of the substrate analog, FdUMP (D. A. Matthews, K. Appelt and S. J. Oatley, (1989) Adv. Enz. Reg., 29, 47-60). We have used protein engineering techniques to change this amino acid residue to valine. This alteration leads to large reductions in the ability of the enzyme to form covalent complexes with substrate (dUMP) or inhibitor (FdUMP) and at least a 100-fold reduction in catalytic activity. These observations show that this arginine plays an important role in maintaining catalytic activity of the enzyme.     

The conserved arginine 380 of Hsp90 is not a catalytic residue, but stabilizes the closed conformation required for ATP hydrolysis

Hsp90, a dimeric ATP-dependent molecular chaperone, is required for the folding and activation of numerous essential substrate "client" proteins including nuclear receptors, cell cycle kinases, and telomerase. Fundamental to its mechanism is an ensemble of dramatically different conformational states that result from nucleotide binding and hydrolysis and distinct sets of interdomain interactions. Previous structural and biochemical work identified a conserved arginine residue (R380 in yeast) in the Hsp90 middle domain (MD) that is required for wild type hydrolysis activity in yeast, and hence proposed to be a catalytic residue. As part of our investigations on the origins of species-specific differences in Hsp90 conformational dynamics we probed the role of this MD arginine in bacterial, yeast, and human Hsp90s using a combination of structural and functional approaches. While the R380A mutation compromised ATPase activity in all three homologs, the impact on ATPase activity was both variable and much more modest (2-7 fold) than the mutation of an active site glutamate (40 fold) known to be required for hydrolysis. Single particle electron microscopy and small-angle X-ray scattering revealed that, for all Hsp90s, mutation of this arginine abrogated the ability to form the closed "ATP" conformational state in response to AMPPNP binding. Taken together with previous mutagenesis data exploring intra- and intermonomer interactions, these new data suggest that R380 does not directly participate in the hydrolysis reaction as a catalytic residue, but instead acts as an ATP-sensor to stabilize an NTD-MD conformation required for efficient ATP hydrolysis.     

The 1.62 A structure of Thermoascus aurantiacus endoglucanase: completing the structural picture of subfamilies in glycoside hydrolase family 5

The crystal structure of Thermoascus aurantiacus endoglucanase (Cel5A), a family 5 glycoside hydrolase, has been determined to 1.62 A resolution by multiple isomorphous replacement with anomalous scattering. It is the first report of a structure in the subfamily to which Cel5A belongs. Cel5A consists solely of a catalytic module with compact eight-fold beta/alpha barrel architecture. The length of the tryptophan-rich substrate binding groove suggests the presence of substrate binding subsites -4 to +3. Structural comparison shows that two glycines are completely conserved in the family, in addition to the two catalytic glutamates and six other conserved residues previously identified. Gly 44 in particular is part of a type IV C-terminal helix capping motif, whose disruption is likely to affect the position of an essential conserved arginine. One aromatic residue (Trp 170 in Cel5A), not conserved in term of sequence, is nonetheless spatially conserved in the substrate binding groove. Its role might be to force the bend that occurs in the polysaccharide chain on binding, thus favoring substrate distortion at subsite -1.     

Role of a conserved arginine residue during catalysis in serine palmitoyltransferase

All sphingolipid-producing organisms require the pyridoxal 5'-phosphate (PLP)-dependent serine palmitoyltransferase (SPT) to catalyse the first reaction on the de novo sphingolipid biosynthetic pathway. SPT is a member of the alpha oxoamine synthase (AOS) family that catalyses a Claisen-like condensation of palmitoyl-CoA and L-serine to form 3-ketodihydrosphingosine (KDS). Protein sequence alignment across various species reveals an arginine residue, not involved in PLP binding, to be strictly conserved in all prokaryotic SPTs, the lcb2 subunits of eukaryotic SPTs and all members of the AOS family. Here we use UV-vis spectroscopy and site-directed mutagenesis, in combination with a substrate analogue, to show that the equivalent residue (R370) in the SPT from Sphingomonas wittichii is required to form the key PLP:L-serine quinonoid intermediate that condenses with palmitoyl-CoA and thus plays an essential role enzyme catalysis.     

Relevant Double Mutations in Bioengineered Streptomyces clavuligerus Deacetoxycephalosporin C Synthase Result in Higher Binding Specificities Which Improve Penicillin Bioconversion

Streptomyces clavuligerus deacetoxycephalosporin C synthase (ScDAOCS) is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid, which is a precursor for cephalosporin synthesis. Single mutations of six amino acid residues, V275, C281, N304, I305, R306, and R307, were previously shown to result in enhanced levels of ampicillin conversion, with activities ranging from 129 to 346% of the wild-type activity. In this study, these mutations were paired to investigate their effects on enzyme catalysis. The bioassay results showed that the C-terminal mutations (N304X [where X is alanine, leucine, methionine, lysine, or arginine], I305M, R306L, and R307L) in combination with C281Y substantially increased the conversion of ampicillin; the activity was up to 491% of the wild-type activity. Similar improvements were observed for converting carbenicillin (up to 1,347% of the wild-type activity) and phenethicillin (up to 1,109% of the wild-type activity). Interestingly, the N304X R306L double mutants exhibited lower activities for penicillin G conversion, and activities that were 40 to 114% of wild-type enzyme activity were detected. Based on kinetic studies using ampicillin, it was clear that the increases in the activities of the double mutants relative to those of the corresponding single mutants were due to enhanced substrate binding affinities. These results also validated the finding that the N304R and I305M mutations are ideal for increasing the substrate binding affinity and turnover rate of the enzyme, respectively. This study provided further insight into the structure-function interaction of ScDAOCS with different penicillin substrates, thus providing a useful platform for further rational modification of its enzymatic properties.     

Interaction of the FimB integrase with the fimS invertible DNA element in Escherichia coli in vivo and in vitro

The FimB protein is a site-specific recombinase that inverts the fimS genetic switch in Escherichia coli. Based on amino acid sequence analysis alone, FimB has been assigned to the integrase family of tyrosine recombinases. We show that amino acid substitutions at positions R47, H141, R144, and Y176, corresponding to highly conserved members of the catalytic motif of integrase proteins, render FimB incapable of inverting the fimS element in vivo. The arginine substitutions reduced the ability of FimB to bind to fimS in vivo or in vitro, while the substitution R144Q resulted in a protein unable to bind independently to the half sites located at the left end of fimS in phase-on bacteria. These data confirm that FimB is an integrase and suggest that residue R144 has a role in binding to a specific component of the fim switch.     

The role of conserved arginine residue in loop 4 of glycoside hydrolase family 10 xylanases

An arginine residue in loop 4 connecting beta strand 4 and alpha-helix 4 is conserved in glycoside hydrolase family 10 (GH10) xylanases. The arginine residues, Arg(204) in xylanase A from Bacillus halodurans C-125 (XynA) and Arg(196) in xylanase B from Clostridium stercorarium F9 (XynB), were replaced by glutamic acid, lysine, or glutamine residues (XynA R204E, K and Q, and XynB R196E, K and Q). The pH-k(cat)/K(m) and the pH-k(cat) relationships of these mutant enzymes were measured. The pK(e2) and pK(es2) values calculated from these curves were 8.59 and 8.29 (R204E), 8.59 and 8.10 (R204K), 8.61 and 8.19 (R204Q), 7.42 and 7.19 (R196E), 7.49 and 7.18 (R196K), and 7.86 and 7.38 (R196Q) respectively. Only the pK(es2) value of arginine derivatives was less than those of the wild types (8.49 and 9.39 [XynA] and 7.62 and 7.82 [XynB]). These results suggest that the conserved arginine residue in GH10 xylanases increases the pK(a) value of the proton donor Glu during substrate binding. The arginine residue is considered to clamp the proton donor and subsite +1 to prevent structural change during substrate binding.     

Arginines 29 and 59 of elongation factor G are important for GTP hydrolysis or translocation on the ribosome

GTP hydrolysis by elongation factor G (EF-G) is essential for the translocation step in protein elongation. The low intrinsic GTPase activity of EF-G is strongly stimulated by the ribosome. Here we show that a conserved arginine, R29, of Escherichia coli EF-G is crucial for GTP hydrolysis on the ribosome, but not for GTP binding or ribosome interaction, suggesting that it may be directly involved in catalysis. Another conserved arginine, R59, which is homologous to the catalytic arginine of G(alpha) proteins, is not essential for GTP hydrolysis, but influences ribosome binding and translocation. These results indicate that EF-G is similar to other GTPases in that an arginine residue is required for GTP hydrolysis, although the structural changes leading to GTPase activation are different.