The role of hydrophobic active‐site residues in substrate specificity and acyl transfer activity of penicillin acylase

Abstract

Penicillin acylase of Escherichia coli catalyses the hydrolysis and synthesis of b-lactam antibiotics. To study the role of hydrophobic residues in these reactions, we have mutated three active-site phenylalanines. Mutation of aF146, bF24 and bF57 to Tyr, Trp, Ala or Leu yielded mutants that were still capable of hydrolysing the chromogenic substrate 2-nitro-5-[(phenylacetyl)amino]-benzoic acid. Mutations on positions aF146 and bF24 influenced both the hydrolytic and acyl transfer activity. This caused changes in the transferase/hydrolase ratios, ranging from a 40-fold decrease for aF146Y and aF146W to a threefold increase for aF146L and bF24A, using 6-aminopenicillanic acid as the nucleophile. Further analysis of the bF24A mutant showed that it had specificity constants (k cat /K m ) for p-hydroxyphenylglycine methyl ester and phenylglycine methyl ester that were similar to the wild-type values, whereas the specificity constants for p-hydroxyphenylglycine amide and phenylglycine amide had decreased 10-fold, due to a decreased k cat value. A low amidase activity was also observed for the semisynthetic penicillins amoxicillin and ampicillin and the cephalosporins cefadroxil and cephalexin, for which the k cat values were fivefold to 10-fold lower than the wild-type values. The reduced specificity for the product and the high initial transferase/hydrolase ratio of bF24A resulted in high yields in acyl transfer reactions.Keywords: site-directed mutagenesis; b-lactam antibiotics; penicillin acylase; substrate specificity; transferase/ hydrolase ratio.Penicillin acylase (PA) of Escherichia coli (EC 3.5.1.11) catalyses the hydrolysis of penicillin G to phenylacetic acid (PAA) and 6-aminopenicillanic acid (6-APA). PA is a heterodimeric periplasmic protein consisting of a small a subunit and a large b subunit, which are formed by processing of a precursor protein. The catalytic nucleophile, a serine, is located at the N-terminus, which is a hallmark of the family of N-terminal nucleophile (Ntn) hydrolases, a class of enzymes which share a common fold around the active site and contain a catalytic serine, cysteine or threonine at the N-terminal position [1]. The reaction mechanism of PA involves the formation of a covalent intermediate and is similar to the well-known mechanism of serine proteases. After attack on the carbonyl carbon of the amide bond by the active-site nucleophile, a covalent acyl-enzyme is formed via a tetrahedral transition state in which the negatively charged oxyanion is stabilized by H-bonds to the oxyanion hole residues bN241 and bA69 [2]. After expulsion of the leaving group from the active site, the acyl-enzyme is deacylated by H 2 O or another nucleophile, yielding the final transacylation product and the free enzyme.PA is used for the production of 6-aminopenicillanic acid (6-APA) by the hydrolysis of penicillin G, but can also be used for the production of semisynthetic b-lactam antibiotics, in which the enzyme catalyses the condensation of an acyl group and a 6-APA molecule [3]. In this cond...

Acetobacter turbidans α-Amino Acid Ester Hydrolase

“…Apparently, the mutation affects the interaction of the acyl enzyme with the competing nucleophilic acyl acceptors, water and the ␤-lactam nucleus, by altering the way in which the acyl enzyme is presented to an acyl acceptor, or the way in which the acceptor is bound and/or activated. Possibly, the geometry of the atoms around the scissile bond between enzyme and acyl group is changed to favor attack by a ␤-lactam rather than by water (26). An alternative explanation is that Tyr 112 participates directly in binding the acceptor, in which case a change in the position of Tyr 112 could affect both binding affinity and binding mode.…”

Improvement of Catalytic Properties of Escherichia coli Penicillin G Acylase Immobilized on Glyoxyl Agarose by Addition of a Six-Amino-Acid Tag

“…The periplasmic extract (3) was purified on a Q-Sepharose column (Pharmacia), with a 0-to-75 mM NaCl gradient in 50 mM Tris HCl (pH 8.5) (average yield was 18 mg of purified protein per 30 g of wet cells). pET28-3G3KPAC was expressed in BL21 Star(DE3) cells as described previously, except that the temperature for the induction of expression was lowered to 20°C, due to the increased sensitivity and lower yield of processing and maturation (2,4,8,13,18,19) of the modified enzyme (data not shown). The periplasmic extract was precipitated with 75% (NH 4 ) 2 SO 4 and the pellet, resuspended in 1 M (NH 4 ) 2 SO 4 and 50 mM potassium phosphate (pH 7), was purified on a phenyl Sepharose column with a gradient of 0 to 30% ethylene glycol in 50 mM potassium phosphate (pH 7).…”

Engineering the Substrate Specificity of a Thermophilic Penicillin Acylase from Thermus thermophilus

“…Residues ␣Phe146, ␤Phe24, and ␤Phe57 have been described to determine the acyl specificity in EcoPGA (20). However, as shown in Fig.…”

“…Among them, PGAs are the most abundant enzymes (e.g., from E. coli [35], Kluyvera cryocescens, Kluyvera citrophila [36], Arthrobacter viscosus [37], and A. faecalis [34]). These enzymes exhibit a narrow substrate specificity and are unable to hydrolyze aliphatic penicillins due to the aromatic nature of their acyl binding sites (20). The penicillin V acylases from Bacillus sphaericus (38), Bacillus subtilis, and Fusarium sp.…”