The binding of penicillin to penicillin acylase was studied by X-ray crystallography. The structure of the enzyme-substrate complex was determined after soaking crystals of an inactive betaN241A penicillin acylase mutant with penicillin G. Binding of the substrate induces a conformational change, in which the side chains of alphaF146 and alphaR145 move away from the active site, which allows the enzyme to accommodate penicillin G. In the resulting structure, the beta-lactam binding site is formed by the side chains of alphaF146 and betaF71, which have van der Waals interactions with the thiazolidine ring of penicillin G and the side chain of alphaR145 that is connected to the carboxylate group of the ligand by means of hydrogen bonding via two water molecules. The backbone oxygen of betaQ23 forms a hydrogen bond with the carbonyl oxygen of the phenylacetic acid moiety through a bridging water molecule. Kinetic studies revealed that the site-directed mutants alphaF146Y, alphaF146A and alphaF146L all show significant changes in their interaction with the beta-lactam substrates as compared with the wild type. The alphaF146Y mutant had the same affinity for 6-aminopenicillanic acid as the wild-type enzyme, but was not able to synthesize penicillin G from phenylacetamide and 6-aminopenicillanic acid. The alphaF146L and alphaF146A enzymes had a 3-5-fold decreased affinity for 6-aminopenicillanic acid, but synthesized penicillin G more efficiently than the wild type. The combined results of the structural and kinetic studies show the importance of alphaF146 in the beta-lactam binding site and provide leads for engineering mutants with improved synthetic properties.

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...

In hepatocytes, cAMP/PKA activity stimulates the exocytic insertion of apical proteins and lipids and the biogenesis of bile canalicular plasma membranes. Here, we show that the displacement of PKA-RIIalpha from the Golgi apparatus severely delays the trafficking of the bile canalicular protein MDR1 (P-glycoprotein), but not that of MRP2 (cMOAT), DPP IV and 5'NT, to newly formed apical surfaces. In addition, the direct trafficking of de novo synthesized glycosphingolipid analogues from the Golgi apparatus to the apical surface is inhibited. Instead, newly synthesized glucosylceramide analogues are rerouted to the basolateral surface via a vesicular pathway, from where they are subsequently endocytosed and delivered to the apical surface via transcytosis. Treatment of HepG2 cells with the glucosylceramide synthase inhibitor PDMP delays the appearance of MDR1, but not MRP2, DPP IV, and 5'NT at newly formed apical surfaces, implicating glucosylceramide synthesis as an important parameter for the efficient Golgi-to-apical surface transport of MDR1. Neither PKA-RIIalpha displacement nor PDMP inhibited (cAMP-stimulated) apical plasma membrane biogenesis per se, suggesting that other cAMP effectors may play a role in canalicular development. Taken together, our data implicate the involvement of PKA-RIIalpha anchoring in the efficient direct apical targeting of distinct proteins and glycosphingolipids to newly formed apical plasma membrane domains and suggest that rerouting of Golgi-derived glycosphingolipids may underlie the delayed Golgi-to-apical surface transport of MDR1.

Halohydrin dehalogenase obtained from Agrobacterium radiobacter AD1, has been tested for the nitrite-mediated ring opening of epoxides. This reaction mainly leads to the formation of unstable hydroxynitrite ester intermediates, which can be further hydrolyzed to the corresponding diols. This conversion proceeds with high enantioselectivity and high regioselectivity towards styrene oxide derivatives. It has been concluded that halohydrin dehalogenase can serve as an attractive alternative to epoxide hydrolases in the preparation of enantiopure epoxides by kinetic resolution.