Comparative characterization of new glutaryl 7-ACA and cephalosporin C acylases

Aramori, Ichiro; Fukagawa, Masao; Tsumura, Mana; Iwami, Morita; Ono, Hiroki; Ishitani, Yosuke; Kojo, Hitoshi; Kohsaka, Masanobu; Ueda, Yoshio; Imanaka, Hiroshi

doi:10.1016/0922-338x(92)90158-q

Cited by 37 publications

(14 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The optimal pH for DthA was 7.9, which is similar to the pH 8 optimum of prolyl oligopeptidase and another homolog of DthA, the glutaryl 7‐amino cephalosporanic acid acylase from Bacillus laterosporus J1 [23,24], but much higher than the pH optima for both XcAEH and AtAEH (pH 6.4 and pH 6, respectively) [25,26]. The unusually low pH optima of the AEHs is attributed to their preferential binding to the charged form of their substrates, which contain an amino group with a p K a of ∼ 7 [25].…”

Section: Discussionmentioning

confidence: 94%

Kinetics of hydrolysis and mutational analysis of N,N‐diethyl‐m‐toluamide hydrolase from Pseudomonas putida DTB

Rivera-Cancel

Sanders

2012

The FEBS Journal

View full text Add to dashboard Cite

The initial step in the biodegradation pathway of N,N-diethyl-m-toluamide (DEET) in Pseudomonas putida strain DTB is catalyzed by DEET hydrolase (DthA), which hydrolyzes the amide bond to yield 3-methylbenzoic acid and diethylamine. In order to extend our understanding of DthA, the enzyme was purified and characterized. The enzyme is most active at pH 7.9, and is probably a tetramer in its native state. The kinetic parameters of the wild-type enzyme are K m = 10.2 ± 0.8 lM, k cat = 5.53 ± 0.09 s )1 , andMild substrate inhibition was observed with DEET concentrations over 500 lM. A homology model of DthA was used to guide mutational analysis of the active site, confirming that the catalytic triad is formed by Ser166, Ap292, and His320. The oxyanion hole is formed by the side chain OH of Tyr84 and the backbone amide of Trp167, with the Tyr84 OH being essential for enzyme activity. The DthA model also revealed a hydrophobic substrate-binding pocket comprosed of Trp167, Met170, and Trp214. W167A and M170A mutations decreased enzymatic activity and exacerbated substrate inhibition, whereas Trp214, which probably plays a role in substrate recognition, was essential for enzymatic activity. The pH rate profile of DthA was fitted to two ionizable groups (pK a1 = 6.1 and pK a2 = 9.9) that probably correspond to Ne of His320 and the OH of Tyr84, respectively. In addition to catalyzing the hydrolysis of DEET, DthA hydrolyzed a variety of esters and amides.

show abstract

Section: Discussionmentioning

confidence: 94%

Kinetics of hydrolysis and mutational analysis of N,N‐diethyl‐m‐toluamide hydrolase from Pseudomonas putida DTB

Rivera-Cancel

Sanders

2012

The FEBS Journal

View full text Add to dashboard Cite

show abstract

“…It was predicted that the N‐terminal residue Ser of the β‐subunits of CAI, CAII and CAIII, similar to that of PGA, might act as a nucleophile in catalysis, although no experimental results had been provided [5,24,37]. In PGA, Ser could be replaced by Cys for processing but not for activity [38].…”

Section: Discussionmentioning

confidence: 99%

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

Chen

Jiang³

et al. 1999

European Journal of Biochemistry

View full text Add to dashboard Cite

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 a-and b-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 b-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.

show abstract

“…and also shows glutarylcephalosporanic acid acylase activity which is 25-fold higher than that against cephalosporin C (Aramori et al, 1992). In previous studies, Tyr270 in N176 acylase was found to be the residue that was specifically modified both with an affinity-label reagent, 7P-(6-bromohexanoy1amido)cephalosporanic acid (Ishii et al, 1994) and with a non-specific nitration reagent, tetranitromethane (Nobbs et al, 1994).…”

mentioning

confidence: 99%

“…The assay of glutarylcephalosporanic acid acylase activity is described in Materials and Methods. The expression level with E. coli JM109/pCCN176-3 ( Aramori et al, 1992) was 0.5 units/ml. The specific activity of the wild-type enzyme against glutarylcephalosporanic acid (46.3 units/mg protein) is defined as 100.…”

mentioning

confidence: 99%

High‐level Production, Chemical Modification and Site‐directed Mutagenesis of a Cephalosporin C Acylase from Pseudomonas Strain N176

Ishii

Saito

Fujimura

et al. 1995

European Journal of Biochemistry

View full text Add to dashboard Cite

A cephalosporin acylase from Pseudomonas strain N176 hydrolyses both 7-p-(4-~arboxybutanamido)-cephalosporanic acid (glutarylcephalosporanic acid) and cephalosporin C to 7-amino-cephalosporanic acid. However, its productivity in the original host was low and its activity against cephalosporin C was not sufficient for direct large-scale production of 7-amino-cephalosporanic acid. In order to overcome these problems, we established a high-level expression system for the acylase in Escherichia coli. Tyr270 in the acylase is reported to play an important role in the interaction with glutarylcephalosporanic acid, as determined from the reaction with an affinity-label reagent, 7p-(6-bromohexanoylamido) cephalosporanic acid [Ishii, Y., Saito, Y., Sasaki, H., Uchiyama Ferment. Bioeng. 77,, From carbamoylation with potassium cyanate and site-directed point mutagenesis of the cephalosporin C acylase, we have deduced that Tyr270 exists at a position where it can interact with a residue (possibly Ser239) corresponding to inactivation by carbamoylation. We mutated Met269 and Ala271 of the acylase and found that mutation of Met269 to Tyr or Phe caused a 1.6-fold and 1.7-fold increase, respectively, of specific activity against cephalosporin C as compared to that of the wild-type enzyme. Kinetic studies of these mutants revealed that their k,,, values increased, although their Km values against cephalosporin C were not changed. These data indicate that the mutation of Met269 near Tyr270 induces a minor conformational change to increase the stability of the activated complex with the enzyme and cephalosporin C. In particular, a mutant in which Met269 was replaced by Tyr was 2.5-fold more efficient in converting cephalosporin C to 7-aminocephalosporanic acid than the wild-type enzyme under conditions similar to those in a bio-reactor system.Keywords. Cephalosporin C ; site-directed mutagenesis ; 7-amino-cephalosporanic acid ; carbamoylation ; bioreactor.Recently, enzymic production of chemicals has been preferred to that of the chemical method because plant investment costs are lowered and the ecological problem of organic solvent disposal is eliminated. For 7-amino-cephalosporanic acid, a key intermediate in the production of cephem antibiotics, a two-step enzymic method is attractive in which cephalosporin C is converted to 7-~-(4-carboxybutanamido)cephalosporanic acid (glutarylcephalosporanic acid) with D-aminO acid oxidase and hydrogen peroxide, followed by hydrolysis of glutarylcephalosporanic acid to 7-amino-cephalosporanic acid with glutarylcephalosporanic acid acylase. Recently, a cephalosporin C acylase which directly catalyzes the hydrolysis of cephalosporin C to 7-amino-cephalosporanic acid and a-amino-adipic acid was isolated from a strain of Pseudomonas sp. N176 (Aramori et al., 1991a). It appeared to be useful for the one-step enzymic production of 7-amino-cephalosporanic acid from cephalosporin C. This acylase, designated as N176 acylase, consists of two polypeptide chains (a and p chains) which are formed from ...

show abstract

Comparative characterization of new glutaryl 7-ACA and cephalosporin C acylases

Cited by 37 publications

References 17 publications

Kinetics of hydrolysis and mutational analysis of N,N‐diethyl‐m‐toluamide hydrolase from Pseudomonas putida DTB

Kinetics of hydrolysis and mutational analysis of N,N‐diethyl‐m‐toluamide hydrolase from Pseudomonas putida DTB

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

High‐level Production, Chemical Modification and Site‐directed Mutagenesis of a Cephalosporin C Acylase from Pseudomonas Strain N176

Contact Info

Product

Resources

About