Publications

• Characterization of an industrial biocatalyst: immobilized glutaryl-7-ACA acylase.

  Monti D., Carrea G., Riva S., Baldaro E., Frare G.

  A batch of the immobilized industrial biocatalyst glutaryl-7-ACA acylase (GA), one of the two enzymes involved in the biotransformation of cephalosporin C (CefC) into 7-aminocephalosporanic acid (7-ACA), was characterized. K(m) value for glutaryl-7-ACA was 5 mM. Enzyme activity was found to be opt ... >> More

  A batch of the immobilized industrial biocatalyst glutaryl-7-ACA acylase (GA), one of the two enzymes involved in the biotransformation of cephalosporin C (CefC) into 7-aminocephalosporanic acid (7-ACA), was characterized. K(m) value for glutaryl-7-ACA was 5 mM. Enzyme activity was found to be optimal at pH between 7 and 9.5 and to increase with temperature and in buffered solutions. To avoid product degradation, optimal reaction conditions were obtained working at 25 degrees C using a 50-mM phosphate buffer, pH 8.0. Immobilized GA showed good stability at pH value below 9 and at temperature up to 30 degrees C. The inactivation of immobilized GA in the presence of different amounts of H(2)O(2), a side product that might be present in the plant-scale industrial solutions of glutaryl-7-ACA, was also investigated, but the deactivation rates were negligible at H(2)O(2) concentration that might be reached under operative conditions. Finally, biocatalyst performance in the complete two-step enzymatic conversion process from CefC to 7-ACA was determined on a laboratory scale. Following the complete conversion of a 75 mM solution of CefC into glutaryl-7-ACA catalyzed by an immobilized D-amino acid oxidase (DAAO), immobilized GA was used for the transformation of this intermediate into the final product 7-ACA. This reaction was repeated for 42 cycles. An estimation of the residual activity of the biocatalyst showed that 50% inactivation of immobilized GA was reached after approximately 300 cycles, corresponding to an enzyme consumption of 0.4 kU per kg of isolated 7-ACA. << Less

  Biotechnol Bioeng 70:239-244(2000) [PubMed] [EuropePMC]

• Affinity alkylation of the Trp-B4 residue of the beta -subunit of the glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130.

  Huang X., Zeng R., Ding X., Mao X., Ding Y., Rao Z., Xie Y., Jiang W., Zhao G.

  Glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130 (C130) was irreversibly inhibited in a time-dependent manner by two substrate analogs bearing side chains of variable length, namely 7beta-bromoacetyl aminocephalosporanic acid (BA-7-ACA) and 7beta-3-bromopropionyl aminocephalospo ... >> More

  Glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130 (C130) was irreversibly inhibited in a time-dependent manner by two substrate analogs bearing side chains of variable length, namely 7beta-bromoacetyl aminocephalosporanic acid (BA-7-ACA) and 7beta-3-bromopropionyl aminocephalosporanic acid (BP-7-ACA). The inhibition of the enzyme with BA-7-ACA was attributable to reaction with a single amino acid residue within the beta-subunit proven by comparative matrix assisted laser desorption/ionization-time of flight mass spectrometry. Further mass spectrometric analysis demonstrated that the fourth tryptophan residue of the beta-subunit, Trp-B4, was alkylated by BA-7-ACA. By (1)H-(13)C HSQC spectroscopy of C130 labeled by BA-2-(13)C-7-ACA, it was shown that tryptophan residue(s) in the enzyme was alkylated, forming a carbon-carbon bond. Replacing Trp-B4 with other amino acid residues caused increases in K(m), decreases in k(cat), and instability of enzyme activity. None of the mutant enzymes except W-B4Y could be affinity-alkylated, but all were competitively inhibited by BA-7-ACA. Kinetic studies revealed that both BA-7-ACA and BP-7-ACA could specifically alkylate Trp-B4 of C130 as well as Tyr-B4 of the mutant W-B4Y. Because these alkylations were energy-requiring under physiological conditions, it is likely that the affinity labeling reactions were catalyzed by the C130 enzyme itself. The Trp-B4 residue is located in the middle of a characteristic alphabetabetaalpha sandwich structure. Therefore, a large conformational alteration during inhibitor binding and transition state formation is likely and suggests that a major conformational change is induced by substrate binding during the course of catalysis. << Less

  J Biol Chem 277:10256-10264(2002) [PubMed] [EuropePMC]

• Crystal structures of glutaryl 7-aminocephalosporanic acid acylase: insight into autoproteolytic activation.

  Kim J.K., Yang I.S., Rhee S., Dauter Z., Lee Y.S., Park S.S., Kim K.H.

  Glutaryl 7-aminocephalosporanic acid acylase (GCA, EC 3.5.1.11) is a member of N-terminal nucleophile (Ntn) hydrolases. The native enzyme is an (alpha beta)(2) heterotetramer originated from an enzymatically inactive precursor of a single polypeptide. The activation of precursor GCA consists of pr ... >> More

  Glutaryl 7-aminocephalosporanic acid acylase (GCA, EC 3.5.1.11) is a member of N-terminal nucleophile (Ntn) hydrolases. The native enzyme is an (alpha beta)(2) heterotetramer originated from an enzymatically inactive precursor of a single polypeptide. The activation of precursor GCA consists of primary and secondary autoproteolytic cleavages, generating a terminal residue with both a nucleophile and a base and releasing a nine amino acid spacer peptide. We have determined the crystal structures of the recombinant selenomethionyl native and S170A mutant precursor from Pseudomonas sp. strain GK16. Precursor activation is likely triggered by conformational constraints within the spacer peptide, probably inducing a peptide flip. Autoproteolytic site solvent molecules, which have been trapped in a hydrophobic environment by the spacer peptide, may play a role as a general base for nucleophilic attack. The activation results in building up a catalytic triad composed of Ser170/His192/Glu624. However, the triad is not linked to the usual hydroxyl but the free alpha-amino group of the N-terminal serine residue of the native GCA. Mutagenesis and structural data support the notion that the stabilization of a transient hydroxazolidine ring during autoproteolysis would be critical during the N --> O acyl shift. The autoproteolytic activation mechanism for GCA is described. << Less

  Biochemistry 42:4084-4093(2003) [PubMed] [EuropePMC]

• Molecular cloning and structure of the gene for 7 beta-(4-carboxybutanamido)cephalosporanic acid acylase from a Pseudomonas strain.

  Matsuda A., Komatsu K.

  A Pseudomonas strain produced an enzyme capable of deacylating 7 beta-(4-carboxybutanamido)cephalosporanic acid to 7-aminocephalosporanic acid in response to glutaric acid. The gene for the enzyme was cloned within the PstI site of pBR325 as a 7.35-kilobase-pair DNA segment from a mutant of this s ... >> More

  A Pseudomonas strain produced an enzyme capable of deacylating 7 beta-(4-carboxybutanamido)cephalosporanic acid to 7-aminocephalosporanic acid in response to glutaric acid. The gene for the enzyme was cloned within the PstI site of pBR325 as a 7.35-kilobase-pair DNA segment from a mutant of this strain whose enzyme is produced constitutively. The gene expression in the primary clone appeared to be low in Escherichia coli but was significantly enhanced by reducing the size of the initial segment coupled with E. coli promoters. Subsequent subcloning resulted in localization of the gene to a 2.45-kilobase-pair fragment. Three clone-specific polypeptides with molecular weights of ca. 16,000, 54,000, and 70,000 were shown by maxicell analysis. The former two corresponded to the small and large subunits of the purified enzyme from the Pseudomonas strain, and the third polypeptide was suggested to be their precursor. This was supported by DNA sequence study together with amino acid sequencing of the amino terminus of both subunits: the sequences for the small and large subunits were localized contiguously in this order on the structural gene without termination codons between them. The nucleotide sequence also disclosed the presence of a signallike sequence preceding that for the small subunit, consistent with the previous observation that the enzyme might be periplasmic in the Pseudomonas strain. Those results suggest a process for the formation of an active enzyme complex from a precursor through two steps of processing. << Less

  J. Bacteriol. 163:1222-1228(1985) [PubMed] [EuropePMC]

• The 2.0 A crystal structure of cephalosporin acylase.

  Kim Y., Yoon K.-H., Khang Y., Turley S., Hol W.G.J.

  <h4>Background</h4>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 treatmen ... >> More

  <h4>Background</h4>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.<h4>Results</h4>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.<h4>Conclusion</h4>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. << Less

  Structure 8:1059-1068(2000) [PubMed] [EuropePMC]