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.     

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.     

Molecular weight analysis of isopenicillin N synthase by electrospray mass spectrometry

The use of electrospray mass spectrometry as a tool in analytical biochemistry was illustrated by determination of the molecular weights of wildtype and recombinant isopenicillin N synthase (IPNS). The molecular weight of recombinant IPNS produced using an expression system which generated soluble protein was found to be between 38,364 and 38,376 Da, ca 60 mass units higher than that of the wildtype material, consistent with the presence of an additional N-terminal glycine in the former. Observed molecular weights were all ca 70 Da higher than that calculated from sequence information, consistent with the complexion of a partially hydrated iron atom to the enzyme during analysis.     

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.     

High level expression in Escherichia coli of isopenicillin N synthase genes from Flavobacterium and Streptomyces, and recovery of active enzyme from inclusion bodies

A T7 promoter-based vector was used to express the isopenicillin N synthase (IPNS) genes of Flavobacterium sp. 12,154 and Streptomyces jumonjinensis in Escherichia coli. Most of the IPNS synthesized at 37 degrees C, and representing some 22% and 51% of the total cell protein respectively, occurred in an insoluble, enzymatically inactive form. Active IPNS was recovered in a rapid and simple two-step procedure in which the insoluble material was first denatured in 5 M urea and then refolded by passing the solubilized IPNS through a G-25 Sephadex sizing column. Further chromatography on DEAE-Sepharose resulted in highly active IPNS preparations. This procedure was found to be well suited for scaling up to produce large amounts of IPNS.     

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.     

Spontaneous chemical reversion of an active site mutation: deamidation of an asparagine residue replacing the catalytic aspartic acid of glutamate dehydrogenase

A mutant (D165N) of clostridial glutamate dehydrogenase (GDH) in which the catalytic Asp is replaced by Asn surprisingly showed a residual 2% of wild-type activity when purified after expression in Escherichia coli at 37 degrees C. This low-level activity also displayed Michaelis constants for substrates that were remarkably similar to those of the wild-type enzyme. Expression at 8 degrees C gave a mutant enzyme preparation 1000 times less active than the first preparation, but progressively, over 2 weeks' incubation at 37 degrees C in sealed vials, this enzyme regained 90% of the specific activity of wild type. This suggested that the mutant might undergo spontaneous deamidation. Mass spectrometric analysis of tryptic peptides derived from D165N samples treated in various ways showed (i) that the Asn is in place in D165N GDH freshly prepared at 8 degrees C; (ii) that there is a time-dependent reversion of this Asn to Asp over the 2-week incubation period; (iii) that detectable deamidation of other Asn residues, in Asn-Gly sequences, mainly occurred in sample workup rather than during the 2-week incubation; (iv) that there is no significant deamidation of other randomly chosen Asn residues in this mutant over the same period; and (v) that when the protein is denatured before incubation, no deamidation at Asn-165 is detectable. It appears that this deamidation depends on the residual catalytic machinery of the mutated GDH active site. A literature search indicates that this finding is not unique and that Asn may not be a suitable mutational replacement in the assessment of putative catalytic Asp residues by site-directed mutagenesis.     

Characterization and structural modeling of a novel thermostable glycine oxidase from Geobacillus kaustophilus HTA426

Glycine oxidase from Geobacillus kaustophilus HTA426 (GOXK) is a 43 kDa monomer flavoenzyme containing noncovalently bound FAD. The induction of the enzyme resulted in the expression of a fully soluble protein with higher specific activity than those previously reported for GOX from B. subtilis (GOXB). A study of the kinetic properties of this novel GOXK revealed the lowest KM values for most of the substrates analyzed, with the exception of D-proline which kept a similar value and had the highest Vmax value reported. The Vmax/KM ratio maintained a substrate preference of GOXK for amines of small size, like glycine, sarcosine, N-ethyl-glycine, and glycine-ethyl-ester. GOXK presented good stability at 60-70 degrees C and in alkaline media (pH 6-9.5). The putative tridimensional structure was modeled by sequence alignment and by comparing the changes between GOXK and GOXB, and the residues that could be responsible for the substrate specificity as well as those essential for the catalytic activity were found. The comparison between the possible topology of GOXK with that of GOXB showed changes at the putative interactions between monomers for the building of the tetrameric oligomerization.     

High level expression in Escherichia coli of a fungal gene under the control of strong promoters

A recent report (Patino et al., (1989) FEMS Microbiol. Lett. 58, 139-144) described the low level expression, in Escherichia coli, of the Isopenicillin N Synthase (IPNS) gene from Cephalosporium acremonium under the control of strong promoters. We report here our work on the expression of the IPNS gene. Plasmids containing the IPNS gene under the control of the trp or trc promoters directed synthesis of high levels of active IPNS in E. coli. Constitutive and inductive high level IPNS expression systems have been developed. Importantly, the expression vectors do not encode beta-lactamase so IPNS activity can be determined directly by biological assays. Analysis by nmr verified that the IPNS produced from these expression systems catalysed the conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (LLD-ACV) to isopenicillin N in high yield.     

PCR cloning, heterologous expression, and characterization of isopenicillin N synthase from Streptomyces lipmanii NRRL 3584

A key step which involves the cyclization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to the bicyclic ring structure of isopenicillin N in the penicillin and cephalosporin biosynthetic pathway, is catalyzed by isopenicillin N synthase (IPNS). In this study, an IPNS gene from Streptomyces lipmanii NRRL 3584 (slIPNS) was cloned via PCR-based homology cloning, sequenced and expressed in Escherichia coli. Soluble slIPNS was overexpressed up to 21% of total soluble protein, and verified to be functionally active when in an IPNS enzymatic assay. Sequence comparison of the slIPNS gene obtained (excluding the consensus primer sequences) with another cloned IPNS from S. lipmanii 16884.3, revealed one three-nucleotide deletion and three closely-spaced single nucleotide deletions. Furthermore, this paper also reports the first instance of the usage of PCR as an alternative and rapid strategy for IPNS cloning using consensus primers.     

Effect of the active site D25N mutation on the structure, stability, and ligand binding of the mature HIV-1 protease

All aspartic proteases, including retroviral proteases, share the triplet DTG critical for the active site geometry and catalytic function. These residues interact closely in the active, dimeric structure of HIV-1 protease (PR). We have systematically assessed the effect of the D25N mutation on the structure and stability of the mature PR monomer and dimer. The D25N mutation (PR(D25N)) increases the equilibrium dimer dissociation constant by a factor >100-fold (1.3 +/- 0.09 microm) relative to PR. In the absence of inhibitor, NMR studies reveal clear structural differences between PR and PR(D25N) in the relatively mobile P1 loop (residues 79-83) and flap regions, and differential scanning calorimetric analyses show that the mutation lowers the stabilities of both the monomer and dimer folds by 5 and 7.3 degrees C, respectively. Only minimal differences are observed in high resolution crystal structures of PR(D25N) complexed to darunavir (DRV), a potent clinical inhibitor, or a non-hydrolyzable substrate analogue, Ac-Thr-Ile-Nle-r-Nle-Gln-Arg-NH(2) (RPB), as compared with PR.DRV and PR.RPB complexes. Although complexation with RPB stabilizes both dimers, the effect on their T(m) is smaller for PR(D25N) (6.2 degrees C) than for PR (8.7 degrees C). The T(m) of PR(D25N).DRV increases by only 3 degrees C relative to free PR(D25N), as compared with a 22 degrees C increase for PR.DRV, and the mutation increases the ligand dissociation constant of PR(D25N).DRV by a factor of approximately 10(6) relative to PR.DRV. These results suggest that interactions mediated by the catalytic Asp residues make a major contribution to the tight binding of DRV to PR.     

Site-directed mutagenesis of glycine-14 and two "critical" cysteinyl residues in Drosophila alcohol dehydrogenase

Three amino acid residues (glycine-14, cysteine-135, and cysteine-218) previously speculated to be important for the structure and function of Drosophila melanogaster alcohol dehydrogenase have been investigated by using site-directed mutagenesis followed by kinetic analysis and chemical modification. Mutating glycine-14 to valine (G14V) virtually inactivates Drosophila ADH, and substitution of alanine at this position (G14A) causes a 31% decrease in activity. Thermal denaturation and kinetic and inhibition studies further demonstrate that replacing glycine-14 with either alanine or valine leads to structural changes in the NAD binding domain. These results provide direct evidence for the role played by glycine-14 in maintaining the correct conformation in the NAD binding domain. On the other hand, changing of cysteine-135, -218, or both to alanine (C135A, C218A, and C135A/C218A) causes no decrease in the catalytic activity of the enzyme, indicating that neither of the cysteinyl residues is essential for catalysis. C135A and wild-type enzyme are both inactivated by DTNB. In contrast, C218A and C135A/C218A are unaffected by DTNB treatment. DTNB modification of cysteine-218 can be prevented by the substrates NAD and 2-propanol, suggesting that cysteine-218 may be in the vicinity of the active site. Cysteine-135 which is normally insensitive to DTNB becomes accessible in the presence of 2-propanol and/or NAD, suggesting a conformational change induced by binding of these substrates.     

Photoaffinity labelling of isopenicillin N synthetase by laser-flash photolysis.

Isopenicillin N synthetase (IPNS) from Acremonium chrysogenum was photolabelled by laser-flash photolysis in the presence of a diazirinyl-containing substrate, 2-[3-(3-trifluoromethyl-3H-diazirin-3-yl)-phenoxy]acetyl-S- methyloxycarbonylsulphenyl-L-cysteinyl-D-valine (DCV). Labelling of IPNS by DCV is partially inhibited in the presence of an excess of L-alpha-aminoadipoyl-L-cysteinyl-D-valine (ACV), the natural substrate. In the absence of light, DCV is converted into the corresponding penicillin with comparable Km but significantly depressed Vmax relative to ACV. Selective incorporation of [14C]DCV into IPNS has been demonstrated by fluorography of IPNS analysed by SDS/polyacrylamide-gel electrophoresis. Scintillation counting of labelled IPNS purified on an ion-exchange f.p.l.c. column confirms this result. This methodology may be applicable for studies aimed at investigating the binding of substrates to IPNS.     

Overexpression of a recombinant wild-type and His-tagged Bacillus subtilis glycine oxidase in Escherichia coli.

We have cloned the gene coding for the Bacillus subtilis glycine oxidase (GO), a new flavoprotein that oxidizes glycine and sarcosine to the corresponding alpha-keto acid, ammonia and hydrogen peroxide. By inserting the DNA encoding for GO into the multiple cloning site of the expression vector pT7.7 we produced a recombinant plasmid (pT7-GO). The pT7-GO encodes a fully active fusion protein with six additional residues at the N-terminus of GO (MARIRA). In BL21(DE3)pLysS Escherichia coli cells, and under optimal isopropyl thio-beta-D-galactoside induction conditions, soluble and active chimeric GO was expressed up to 1.14 U g(-1) of cell (and a fermentation yield of 3.82 U x L(-1) of fermentation broth). An N-terminal His-tagged protein (HisGO) was also successfully expressed in E. coli as a soluble protein and a fully active holoenzyme. HisGO represents approximately 3.9% of the total soluble protein content of the cell. The His-tagged GO was purified in a single step by nickel-chelate chromatography to a specific activity of 1.06 U x mg(-1) protein at 25 degrees C and with a yield of 98%. The characterization of the purified enzyme showed that GO is a homotetramer of approximately 180 kDa with the spectral properties typical of flavoproteins. GO exhibits good thermal stability, with a Tm of 46 degrees C after 30 min incubation; its stability is maximal in the 7.0-8.5 pH range. A comparison of amino-acid sequence and substrate specificity indicates that GO has similarities to other flavoenzymes acting on primary amines and on D-amino acids.     

Is glycine a surrogate for a d‐amino acid in the collagen triple helix?

Collagen is the most abundant protein in animals. Every third residue in a collagen strand is a glycine with phi, psi = -70 degrees, 175 degrees. A recent computational study suggested that replacing these glycine residues with D-alanine or D-serine would stabilize the collagen triple helix. This hypothesis is of substantial importance, as the glycine residues in collagen constitute nearly 10% of the amino acid residues in humans. To test this hypothesis, we synthesized a series of collagen mimic peptides that contain one or more D-alanine or D-serine residues replacing the canonical glycine residues. Circular dichroism spectroscopy and thermal denaturation experiments indicated clearly that the substitution of glycine with D-alanine or D-serine greatly disfavors the formation of a triple helix. Host-guest studies also revealed that replacing a single glycine residue with D-alanine is more destabilizing than is its replacement with L-alanine, a substitution that results from a common mutation in patients with collagen-related diseases. These data indicate that the glycine residues in collagen are not a surrogate for a D-amino acid and support the notion that the main-chain torsion angles of a glycine residue in the native structure (especially, phi > 0 degrees ) are critical determinants for its beneficial substitution with a D-amino acid in a protein.