Deacylation activity of cephalosporin acylase to cephalosporin C is improved by changing the side-chain conformations of active-site residues

Semisynthetic cephalosporins are primarily synthesized from 7-aminocephalosporanic acid (7-ACA), mainly by environmentally toxic chemical deacylation of cephalosporin C (CPC). Thus, the enzymatic conversion of CPC to 7-ACA by cephalosporin acylase (CA) would be very interesting. However, CAs use glutaryl-7-ACA (GL-7-ACA) as a primary substrate and the enzymes have low turnover rates for CPC. The active-site residues of a CA were mutagenized to various residues to increase the deacylation activity of CPC, based on the active-site conformation of the CA structure. The aim was to generate sterically favored conformation of the active-site to accommodate the D-alpha-aminoadipyl moiety of CPC, the side-chain moiety that corresponds to the glutaryl moiety of GL-7-ACA. A triple mutant of the CA, Q50betaM/Y149alphaK/F177betaG, showed the greatest improvement of deacylation activity to CPC up to 790% of the wild-type. Our current study is an efficient method for improving the deacylation activity to CPC by employing the structure-based repetitive saturation mutagenesis.     

Cephalosporin C acylase: dream and(/or) reality

Cephalosporins currently constitute the most widely prescribed class of antibiotics and are used to treat diseases caused by both Gram-positive and Gram-negative bacteria. Cephalosporins contain a 7-aminocephalosporanic acid (7-ACA) nucleus which is derived from cephalosporin C (CephC). The 7-ACA nucleus is not sufficiently potent for clinical use; however, a series of highly effective antibiotic agents could be produced by modifying the side chains linked to the 7-ACA nucleus. The industrial production of higher-generation semi-synthetic cephalosporins starts from 7-ACA, which is obtained by deacylation of the naturally occurring antibiotic CephC. CephC can be converted to 7-ACA either chemically or enzymatically using d-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase. Both these methods show limitation, including the production of toxic waste products (chemical process) and the expense (the enzymatic one). In order to circumvent these problems, attempts have been undertaken to design a single-step means of enzymatically converting CephC to 7-ACA in the course of the past 10 years. The most suitable approach is represented by engineering the activity of a known glutaryl-7-aminocephalosporanic acid acylase such that it will bind and deacylate CephC more preferentially over glutaryl-7-aminocephalosporanic acid. Here, we describe the state of the art in the production of an effective and specific CephC acylase.     

Affinity labeled glutaryl-7-amino cephalosporanic acid acylase C130 can hydrolyze the inhibitor during crystallization

7Beta-bromoacetyl amino cephalosporanic acid (BA-7-ACA), an analog of glutaryl-7-amino cephalosporanic acid (GL-7-ACA), can inhibit and specifically alkylate GL-7-ACA acylase (C130) from Pseudomonas sp.130, forming a carbon-carbon bond between BA-7-ACA and the C-2 on indole ring of Trp-beta4 residue of C130. Here we reported that BA-7-ACA labeled C130 (BA-C130) could self-catalyze the hydrolysis of BA-7-ACA during crystallization process. The hydrolysis was confirmed to be a reaction analogous to the one of GL-7-ACA by comparative matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectrometry analysis. BA-C130 was inactive at room temperature, but in the process of crystallization at 18 degrees C it catalyzed the hydrolysis of BA-7-ACA, and thus made the latter become a substrate. Meanwhile, in crystals, 7-ACA was released but the acetic acid still bound with Trp-beta4, and as a result, the enzyme remained to be inactive. These results demonstrated that Trp-beta4 in the alphabetabetaalpha motif was critical and sensitive for the activity of C130 and also suggested that there was a conformational change induced by deacylation during the process of crystallization.     

Directed evolution of a glutaryl acylase into an adipyl acylase.

Semi-synthetic cephalosporin antibiotics belong to the top 10 of most sold drugs, and are produced from 7-aminodesacetoxycephalosporanic acid (7-ADCA). Recently new routes have been developed which allow for the production of adipyl-7-ADCA by a novel fermentation process. To complete the biosynthesis of 7-ADCA a highly active adipyl acylase is needed for deacylation of the adipyl derivative. Such an adipyl acylase can be generated from known glutaryl acylases. The glutaryl acylase of Pseudomonas SY-77 was mutated in a first round by exploration mutagenesis. For selection the mutants were grown on an adipyl substrate. The residues that are important to the adipyl acylase activity were identified, and in a second round saturation mutagenesis of this selected stretch of residues yielded variants with a threefold increased catalytic efficiency. The effect of the mutations could be rationalized on hindsight by the 3D structure of the acylase. In conclusion, the substrate specificity of a dicarboxylic acid acylase was shifted towards adipyl-7-ADCA by a two-step directed evolution strategy. Although derivatives of the substrate were used for selection, mutants retained activity on the beta-lactam substrate. The strategy herein described may be generally applicable to all beta-lactam acylases.     

GL-7ACA酰化酶表达检测系统的建立

戊二酰－7－氨基头孢烷酸（GL－7ACA）酰化酶能够催化GL－7ACA分解生成7－ACA,后者是工业半合成生产头孢类抗菌素所需的重要前体。为了准确地检测GL－7ACA酰化酶及其突变体的表达,本研究通过构建一系列质粒载体,建立了两个简便有效地测定GL－7ACA酰化酶基因acy表达量的系统,从而可对酶的比活力进行定量。我们将两个报告基因,即儿茶酚双加氧酶基因（xylE)和β－半乳糖苷酶基因（lacZ)分别置于acy基因的下游,使之与acy基因共用一个启动子,进行串联表达,各自构成一个多顺反子系统。实验证明,基因融合后的儿茶酚双加氧酶或β－半乳糖苷酶的活力可以间接反映acy的表达量。     

Enzymatic modifications of cephalosporins by cephalosporin acylase and other enzymes

Semisynthetic cephalosporins are important antibacterials in clinical practice. Semisynthetic cephalosporins are manufactured by derivatizing 7-aminocephalosporanic acid (7-ACA) and its desacetylated form. Microbial enzymes such as D-amino acid oxidase, glutaryl-7-ACA acylase and cephalosporin esterase are being used as biocatalysts for the conversion of cephalosporin C (CEPH-C) to 7-ACA and its desacetylated derivatives. Recent developments in the field of enzymatic modifications of cephalosporin with special emphasis on group of enzymes called as cephalosporin acylase is discussed in this review. Aspects related to screening methods, isolation and purification, immobilization, molecular cloning, gene structure and expression and protein engineering of cephalosporin acylases have been covered. Topics pertaining to enzymatic modifications of cephalosporin by D-amino acid oxidase, cephalosporin methoxylase and beta-lactamase are also covered.     

In vivo post-translational processing and subunit reconstitution of cephalosporin acylase from Pseudomonas sp. 130.

Cephalosporin acylases are a group of enzymes that hydrolyze cephalosporin C (CPC) and/or glutaryl 7-amino cephalosporanic acid (GL-7ACA) to produce 7-amino cephalosporanic acid (7-ACA). The acylase from Pseudomonas sp. 130 (CA-130) is highly active on GL-7ACA and glutaryl 7-aminodesacetoxycephalosporanic acid (GL-7ADCA), but much less active on CPC and penicillin G. The gene encoding the enzyme is expressed as a precursor polypeptide consisting of a signal peptide followed by alpha- and beta-subunits, which are separated by a spacer peptide. Removing the signal peptide has little effect on precursor processing or enzyme activity. Substitution of the first residue of the beta-subunit, Ser, results in a complete loss of enzyme activity, and substitution of the last residue of the spacer, Gly, leads to an inactive and unprocessed precursor. The precursor is supposed to be processed autocatalytically, probably intramolecularly. The two subunits of the acylase, which separately are inactive, can generate enzyme activity when coexpressed in Escherichia coli. Data on this and other related acylases indicate that the cephalosporin acylases may belong to a novel class of enzymes (N-terminal nucleophile hydrolases) described recently.     

Enzymatic Modifications of Cephalosporins by Cephalosporin Acylase and Other Enzymes

ABSTRACT

Semisynthetic cephalosporins are important antibacterials in clinical practice. Semisynthetic cephalosporins are manufactured by derivatizing 7-aminocephalosporanic acid (7-ACA) and its desacetylated form. Microbial enzymes such as D-amino acid oxidase, glutaryl-7-ACA acylase and cephalosporin esterase are being used as biocatalysts for the conversion of cephalosporin C (CEPH-C) to 7-ACA and its desacetylated derivatives. Recent developments in the field of enzymatic modifications of cephalosporin with special emphasis on group of enzymes called as cephalosporin acylase is discussed in this review. Aspects related to screening methods, isolation and purification, immobilization, molecular cloning, gene structure and expression and protein engineering of cephalosporin acylases have been covered. Topics pertaining to enzymatic modifications of cephalosporin by D-amino acid oxidase, cephalosporin methoxylase and β -lactamase are also covered.     

Improvement of the glutaryl-7-aminocephalosporanic acid acylase activity of a bacterial gamma-glutamyltranspeptidase

7-Aminocephalosporanic acid (7-ACA) is an important material in the production of semisynthetic cephalosporins, which are the best-selling antibiotics worldwide. 7-ACA is produced from cephalosporin C via glutaryl-7-ACA (GL-7-ACA) by a bioconversion process using d-amino acid oxidase and cephalosporin acylase (or GL-7-ACA acylase). Previous studies demonstrated that a single amino acid substitution, D433N, provided GL-7-ACA acylase activity for gamma-glutamyltranspeptidase (GGT) of Escherichia coli K-12. In this study, based on its three-dimensional structure, residues involved in substrate recognition of E. coli GGT were rationally mutagenized, and effective mutations were then combined. A novel screening method, activity staining followed by a GL-7-ACA acylase assay with whole cells, was developed, and it enabled us to obtain mutant enzymes with enhanced GL-7-ACA acylase activity. The best mutant enzyme for catalytic efficiency, with a k(cat)/K(m) value for GL-7-ACA almost 50-fold higher than that of the D433N enzyme, has three amino acid substitutions: D433N, Y444A, and G484A. We also suggest that GGT from Bacillus subtilis 168 can be another source of GL-7-ACA acylase for industrial applications.     

Isolation and Characterization of a Pseudomonas Strain Producing Glutaryl-7-Aminocephalosporanic Acid Acylase

Several screening methods were developed for the selection of Pseudomonas strains capable of hydrolyzing glutaryl-7-aminocephalosporanic acid to 7-aminocephalosporanic acid. An isolate exhibiting high acylase activity, designated BL072, was identified as a strain of Pseudomonas diminuta. It grew optimally at pH 7 to 8 and at a temperature of 32 to 40°C, but acylase activity was highest when the strain was grown at 28°C. Mutants of BL072 were generated by nitrosoguanidine treatment and screened for increased production of glutaryl-7-aminocephalosporanic acid acylase. A superior mutant gave a fourfold increase in acylase titer. The cell-associated acylase had similar activities against various glutaryl-cephems but had undetectable activity against cephalosporin C. This acylase may prove useful for the conversion of cephalosporin C to 7-aminocephalosporanic acid.     

The 2.0 A crystal structure of cephalosporin acylase

BACKGROUND: Semisynthetic cephalosporins are primarily synthesized from 7-aminocephalosporanic acid (7-ACA), which is usually obtained by chemical deacylation of cephalosporin C (CPC). The chemical production of 7-ACA includes, however, several expensive steps and requires thorough treatment of chemical wastes. Therefore, an enzymatic conversion of CPC to 7-ACA by cephalosporin acylase is of great interest. The biggest obstacle preventing this in industrial production is that cephalosporin acylase uses glutaryl-7ACA as a primary substrate and has low substrate specificity for CPC. RESULTS: We have solved the first crystal structure of a cephalosporin acylase from Pseudomonas diminuta at 2.0 A resolution. The overall structure looks like a bowl with two "knobs" consisting of helix- and strand-rich regions, respectively. The active site is mostly formed by the distinctive structural motif of the N-terminal (Ntn) hydrolase superfamily. Superposition of the 61 residue active-site pocket onto that of penicillin G acylase shows an rmsd in Calpha positions of 1.38 A. This indicates structural similarity in the active site between these two enzymes, but their overall structures are elsewhere quite different. CONCLUSION: The substrate binding pocket of the P. diminuta cephalosporin acylase provides detailed insight into the ten key residues responsible for the specificity of the cephalosporin C side chain in four classes of cephalosporin acylases, and it thereby forms a basis for the design of an enzyme with an improved conversion rate of CPC to 7-ACA. The structure also provides structural evidence that four of the five different classes of cephalosporin acylases can be grouped into one family of the Ntn hydrolase superfamily.     

Modifying the substrate specificity of penicillin G acylase to cephalosporin acylase by mutating active-site residues

The penicillin G acylase (PGA) and cephalosporin acylase (CA) families, which are members of the N-terminal (Ntn) hydrolases, are valuable for the production of backbone chemicals like 6-aminopenicillanic acid and 7-aminocephalosporanic acid (7-ACA), which can be used to synthesize semi-synthetic penicillins and cephalosporins, respectively. Regardless of the low sequence similarity between PGA and CA, the structural homologies at their active-sites are very high. However, despite this structural conservation, they catalyze very different substrates. PGA reacts with the hydrophobic aromatic side-chain (the phenylacetyl moiety) of penicillin G (PG), whereas CA targets the hydrophilic linear side-chain (the glutaryl moiety) of glutaryl-7-ACA (GL-7-ACA). These different substrate specificities are likely to be due to differences in the side-chains of the active-site residues. In this study, mutagenesis of active-site residues binding the side-chain moiety of PG changed the substrate specificity of PGA to that of CA. This mutant PGA may constitute an alternative source of engineered enzymes for the industrial production of 7-ACA.     

Isolation and characterization of soil strains producing glutaryl-7-aminocephalosporanic acid acylase

A search was undertaken to screen microorganisms that produce an enzyme capable of deacylating glutaryl-7-aminocephalosporanic acid to 7-aminocephalosporanic acid in soil samples. The screening was carried out by preparing enrichment cultures containing glutaryl-7ACA and cephalosporin C as selective carbon sources. A non-β-lactam model compound, glutaryl-p-nitroanilide, was synthesized as a substrate suitable for the rapid screening of microorganisms isolated from the enrichment cultures. Two isolates exhibiting acylase activity, designated BY7.4 and BY8.1, were identified as strains ofPseudomonas species.Pseudomonas BY8.1 showed higher acylase activity toward Gl-7ACA thanPseudomonas BY7.4. Environmental conditions for the optimal acylase activity ofPseudomonas BY8.1 were shown to be pH 9 and 30°C.     

Screening and characterization of microorganisms with glutaryl-7 ADCA acylase activity

A screening of microorganisms producing glutaryl-7 ADCA acylase, an enzyme able to hydrolyse glutaric acid selectively from glutaryl-3-deacetoxy-7-aminocephalosporanic acid (glutaryl-7 ADCA), has been carried out in soil samples. Five microorganisms expressing acylase activity were isolated and classified as Bacillus cereus, Achromobacter xylosooxidans, Bacillus sp., Pseudomonas sp. and Pseudomonas paucimobilis. The screening was carried out by preparing enrichment cultures containing glutaryl-7-ADCA or cephalosporin C as the selective carbon source. Four model compounds (adipoyl-, glutamyl- and glutaryl-p-nitroanilide and glutarylcoumarin), mimicking the glutaryl-7 ADCA -lactam moiety, were synthesized as substrates suitable for the rapid screening of the microorganisms (2500) isolated from the enrichment cultures. A total of 300 strains were active on the model substrates and only 5 displayed acylase activity on glutaryl-7 ADCA. The fermentation parameters, such as pH and inducer concentration, for the optimal acylase expression and acylase specificity towards the model substrates were different for each strain.     

Construction and application of fusion proteins of d-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase for direct bioconversion of cephalosporin C to 7-aminocephalosporanic acid

Two fusion proteins of D-amino acid oxidase (DAAO) and glutaryl-7-aminocephalosporanic acid acylase (GLA) were designed to simplify the bioconversion process of cephalosporin C to 7-aminocephalosporanic acid (7-ACA), which is conventionally produced in a two-step enzymatic process. Two recombinant plasmids, pET-DLA and pET-ALD, were constructed to express fusion proteins of DAAO-linker-GLA (DLA) and GLA-linker-DAAO (ALD), respectively. When the recombinant plasmids were expressed in E. coli, the fusion protein DLA was not correctly folded and only DAAO activity could be detected. ALD, however, possessed activities of both DAAO and GLA, which directly catalyze the conversion of cephalosporin C into 7-ACA.     

一株产多种β-内酰胺类抗生素酰化酶菌株的筛选

为了从大量的候选菌株中快速筛选头孢菌素酰化酶产生菌,设计并合成了一系列头孢菌素酰化酶的底物类似物。这些酰胺类的底物类似物由二部分组成,一部分为与头孢菌素相同或相似的侧链,另外一部分为发色基团或便于检测的基团。它们被酰化酶水解酰胺键以后可以方便快速的检测,因此用于对大量菌株进行快速筛选。采用这些化合物筛选到6株酰化酶阳性菌株。其中菌株ZH0650能够同时水解GL-7ACA和多个底物类似物。进一步研究表明,该菌至少产生3种酰化酶,AD-NABA酰化酶,青霉素G酰化酶和头孢菌素C酰化酶。我们初步纯化了AD-NABA酰化酶和青霉素G酰化酶,并对头孢菌素C酰化酶的活力进行了鉴定。这是首次报道的可以产生青霉素G酰化酶和头孢菌素酰化酶等多种酰化酶的菌株,具有良好的应用前景。     

D-amino Acid Oxidase as an Industrial Biocatalyst

Biocatalysis driven by D-amino acid oxidase is a significant example of the commercial production of high value-added intermediates using enzyme-based technology. The results of the most recent research on this FAD-dependent catalyst are reported here. In particular, insight is given of how in the past few years the main industrial application of this enzyme, i.e. the stereospecific bioconversion of cephalosporin C to glutaryl-7-amino cephalosporanic acid in the two-step production of 7-amino cephalosporanic acid, has been implemented by improving its production and by engineering of the biocatalyst. The set-up and the optimization of different conditions for carrying out the process under different procedures have also been updated.     

Enhancement of glutaryl-7-aminocephalosporanic acid acylase activity of γ-glutamyltranspeptidase of Bacillus subtilis

Semisynthetic cephalosporins, the best-selling antibiotics worldwide, are derived from 7-aminocephalosporanic acid (7-ACA). Currently, in the pharmaceutical industrie, 7-ACA is mainly produced from cephalosporin C by sequential application of D -amino acid oxidase and cephalosporin acylase. Here we study the potential of industrially amenable enzyme γ-glutamyltranspeptidase from Bacillus subtilis for 7-ACA production, since the wild-type γ-glutamyltranspeptidase of B. subtilis has inherent glutaryl-7-aminocephalosporanic acid acylase activity with a k_cat value of 0.0485 s^-1. Its activity has been enhanced by site directed and random mutagenesis. The k_cat/K_m value was increased to 3.41 s^-1 mM^-1 for a E423Y/E442Q/D445N mutant enzyme and the kcat value was increased to 0.508 s^-1 for a D445G mutant enzyme. Consequently, the catalytic efficiency and the turnover rate were improved up to about 1000-fold and 10-fold, compared with the wildtype γ-glutamyltranspeptidase of B. subtilis.     

D-amino Acid Oxidase as an Industrial Biocatalyst

Biocatalysis driven by D-amino acid oxidase is a significant example of the commercial production of high value-added intermediates using enzyme-based technology. The results of the most recent research on this FAD-dependent catalyst are reported here. In particular, insight is given of how in the past few years the main industrial application of this enzyme, i.e. the stereospecific bioconversion of cephalosporin C to glutaryl-7-amino cephalosporanic acid in the two-step production of 7-amino cephalosporanic acid, has been implemented by improving its production and by engineering of the biocatalyst. The set-up and the optimization of different conditions for carrying out the process under different procedures have also been updated.     

Enzymatic transformation of cephalosporin C to 7-amino-cephalosporanic acid. Part II: Single-step procedure using a coimmobilized enzyme system

The enzymatic transformation of cephalosporin C to 7-amino-cephalosporanic acid (7-ACA) using coimmobilized -aminoacid oxidase (DAAO) and 7-β-(4-carboxybutanamido)cephalosporanic acid acylase (Gl-7-ACA acylase) is reported. The results from the coimmobilization of the two enzymes on different carriers and at different ratios of enzyme activities are described. When an inhibitor of catalase activity, such as NaN₃ or H₂O₂, is present, the conversion rate to 7-ACA is higher, but more by-products are obtained. An optimum ratio of 60:1 between the enzymatic activities of DAAO and Gl-7-ACA acylase in the coimmobilized sample at 0.21 Ug⁻¹ Gl-7-ACA acylase activity was determined. The results of using coimmobilized enzymes and of using a mixture of separately immobilized enzymes in the same process are compared.