11 Abstract Penicillin G acylases (PGAs) belong among enantioselective enzymes catalyzing a hydrolysis of stable amide bond in a broad spectrum of substrates, often having high application potential. PGAEc from Escherichia coli and PGAA from microorganism Achromobacter sp. CCM 4824 were used to catalyze enantioselective hydrolyses of seven selected N-phenylacetylated (N-PhAc) α/β-amino acid racemates. The PGAA showed higher stereoselectivity for three (S) enantiomers: N-PhAc-β-homoleucine, N-PhAc-α-tert- leucine and N-PhAc-β-leucine. We have constructed a homology model of PGAA that was used in molecular docking experiments with the same substrates. In-silico experiments reproduced the data from experimental enzymatic resolutions confirming validity of employed modeling protocol. We employed this protocol to evaluate enantiopreference of PGAA towards seven new substrates with application potential. For five of them, high enantioselectivity of PGAA was predicted for. PGAA was further studied in kinetically controlled syntheses of β-lactam antibiotics (SSBA). The PGAA was significantly more efficient at synthese of ampicillin and amoxicillin (higher S/H ratio and product accumulation) compared with PGAEc . Analogously to prediction of enantioselectivity of PGAA towards new substrates this protocol was applied...

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Molecular modelling strategies for evaluation of enzyme selectivity wereinvestigated with a focus on principles of how molecular interactionscould be evaluated to provide information about selectivity. Althoughmolecular modelling provides tools for evaluation of geometrical andenergy features of molecular systems, no general strategies for evaluationof enzyme selectivity exist. Geometrical analyses can be based uponinspection and reasoning about molecular interactions, which provide aneasily accessible way to gain information, but suffer from the risk of biasput in by the modeller. They can also be based on geometrical features ofmolecular interactions such as bond lengths and hydrogen-bond formation.Energy analyses are appealing for their modeller independenceand for the possibility to predict not only stereopreference, but also itsmagnitude.In this thesis, four examples of enantio- or regioselective serinehydrolase-catalysed reaction systems are presented together with developedmodelling protocols for explanation, prediction or enhancement ofselectivity. Geometrical as well as energy-based methodology were used,and provided an understanding of the structural basis of enzymeselectivity. In total, the protocols were successful in making qualitative explanationsand predictions of stereoselectivity, although quantitative determinationswere not achieved.

The best-studied amidase signature (AS) enzyme is probably fatty acid amide hydrolase (FAAH). Closely related to FAAH is mandelamide hydrolase (MAH), whose substrate specificity and mechanism of catalysis are described in this paper. First, we developed a convenient chromogenic substrate, 4-nitrophenylacetamide, for MAH. The lack of reactivity of MAH with the corresponding ethyl ester confirmed the very limited size of the MAH leaving group site. The reactivity of MAH with 4-nitrophenyl acetate and methyl 4-nitrophenyl carbonate, therefore, suggested formation of an “inverse” acyl-enzyme where the small acyl-group occupies the normal leaving group site. We have interpreted the specificity of MAH for phenylacetamide substrates and small leaving groups in terms of its active site structure, using a homology model based on a FAAH crystal structure. The relevant structural elements were compared with those of FAAH. Phenylmethylboronic acid is a potent inhibitor of MAH (Ki = 27 nM), presumably because it forms a transition state analogue structure with the enzyme. O-Acyl hydroxamates were not irreversible inactivators of MAH but some were found to be transient inhibitors.

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京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12822号 / 論農博第2795号 / 新制||農||1025(附属図書館) / 学位論文||H26||N4817(農学部図書室) / 31309 / 京都大学農学研究科食品生物科学専攻 / (主査)教授 喜多 恵子, 教授 三上 文三, 教授 栗原 達夫 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM