Jun'ichi Oda

Site-directed mutagenesis of putative substrate-binding residues reveals a mechanism controlling the different stereospecificities of two tropinone reductases.

Two tropinone reductases (TRs) constitute a key branch point in the biosynthetic pathway of tropane alkaloids, which are mainly produced in several solanaceous plants. The two TRs share 64% identical amino acid residues and reduce the 3-carbonyl group of a common substrate, tropinone, but they produce distinct alcohol products with different stereospecific configurations. Previous x-ray crystallographic analysis has revealed their highly conserved overall folding, and the modeling of tropinone within the putative substrate-binding sites has suggested that the different stereospecificities may be determined solely by the different binding orientations of tropinone to the enzymes. In this study, we have constructed various mutant TRs, in which putative substrate-binding residues from one TR were substituted with those found in the corresponding positions of the other TR. Substitution of five amino acid residues resulted in an almost complete reversal of stereospecificity, indicating that the different stereospecificities are indeed determined by the binding orientation of tropinone. Detailed kinetic analysis of the mutant enzymes has shown that TR stereospecificity is determined by varying the contributions from electrostatic and hydrophobic interactions and that the present TR structures represent highly evolved forms, in which strict stereospecificities and rapid turnover are accomplished together.

Asymmetric ring opening of cyclic acid anhydrides with lipase in organic solvents

Abstract A lipase (Amano P) catalyzed the asymmetric ring opening of 3-substituted glutaric anhydride with 1-butanol to afford the R half esters having 60–91%ee.

Enantiotopic-group differentiation. Catalytic asymmetric ring-opening of prochiral cyclic acid anhydrides with methanol, using cinchona alkaloids

Asymmetric ring-opening of prochiral acid anhydrides (1) with methanol has been achieved by a catalytic quantity of cinchona alkaloids (2). The product, the optically active half-ester (3), has been subjected to functional-group-selective reduction to give the optically active lactones (5). The reaction rate of the ring-opening and the extent of selectivity are dependent on the nature of the reaction medium, the polarity of solvent, and substrate concentration. By selecting the reaction conditions, an enantiometric excess of up to 70% has been obtained. The kinetic isotope effect and other mechanistic investigations suggest that the reaction proceeds via general-base catalysis by the quinuclidine moiety of the base (2), and that the relative configuration of the C-9 hydroxy group with respect to the C-8 quinuclidine amino function determines the selectivity of the reaction.

Structure of the multifunctional loops in the nonclassical ATP-binding fold of glutathione synthetase

A reagent for affinity labelling of the ATP-binding site in glutathione synthetase captures two flexible glycine-rich loops, revealing their structure.

NADH model reaction. Importance of hydroxy-groups in the asymmetric reduction of ethyl benzoylformate

Ethyl benzoylformate has been reduced with an NADH analogue, N-{(1S)-2-hydroxy-1-[(S)-α-hydroxybenzyl]ethyl}-1-propyl-1,4-dihydronicotinamide, to give (S)-mandelate in 2.9–28.5% e.e. The asymmetric yield was remarkably affected by the amount of magnesium perchlorate present and continuously changed throughout the reaction. The possible functions of hydroxy-groups in the model compound are discussed in the light of the resultant stereochemical outcome.

Chemical Inactivation of Lipase in Organic Solvent : A Lipase from Pseudomonas aeruginosa TE3285 is More Like a Typical Serine Enzyme in an Organic Solvent than in Aqueous Media

A microbial lipase from Pseudomonas aeruginosa TE3285 was treated in anhydrous diisopropyl ether with three kinds of serine-reactive reagents, ethyl p-nitrophenyl methylphosphonate (ENMP), diisopropyl fluorophosphate (DFP), and phenylmethylsulfonyl fluoride (PMSF) to lose its catalytic activity for both transesterification in an organic solvent and ester hydrolysis in aqueous system. In contrast with the facile inactivation in an organic solvent, no or very slow inactivation was observed in an aqueous solution. The lipase was shown to behave more like a typical serine enzyme in an organic solvent than in aqueous solution with regard to the chemical inactivation by serine-reactive reagents. The unique behavior of the lipase in an organic solvent may be associated with inferfacial activation of the lipase, which is one of the most distinct characteristics of the lipase family, and the activiation of lipase could be induced by a hydrophobic interaction with an organic solvent.

Catalytic asymmetric induction from prochiral cyclic acid anhydrides using cinchona alkaloids

Asymmetric ring-opening of prochiral cyclic acid anhydrides (1) with methanol was effected by a catalytic amount of cinchona alkaloids (2) with an enantiomeric excess of up to 70% and the product was converted into optically active lactones.

Mechanism-based inactivation of E. coli γ-glutamylcysteine synthetase by phosphinic acid- and sulfoximine-based transition-state analogues

Abstract Phosphinic acid- and sulfoximine-based transition state analogues having a carboxyl group at the β-carbon to the hetero atom exhibited significantly higher potency as mechanism-based inhibitors of E. coli γ-glutamylcysteine synthetase as compared with l -buthionine -SR- sulfoximine . The enhanced inhibition potency is evidenced by both tight binding of the inhibitor and slow enzyme reactivation.

Highly regioselective ring-opening of α- substituted cyclic acid anhydrides catalyzed by lipase

Abstract Lipase Amano P irreversibly catalyzed a ring-opening of α-substituted cyclic acid anhydrides 1 preferentially at the less hindered carbonyl goup to give monoesters with high regioselectivity.

Searching for amino acid sequence motifs among enzymes: the Enzyme–Reaction Database

Recently we have constructed a database--the Enzyme-Reaction Database--which links a chemical structure to amino acid sequences of enzymes that recognize the chemical structure as their ligand. The total number of enzymes registered in the database is 1103 with 6668 NBRF-PIR entry codes and 1756 chemical compounds. The chemical structures and chemical names for 842 compounds are registered in the Chemical-Structure Database on the MACCS system. For each enzyme, the sequences were divided into clusters, and multiply aligned in each cluster to extract a conserved sequence. A total of 158,781 five-residue-long fragments were constructed from 433 conserved sequences and compared among different clusters of different enzymes. One of these motifs shared by different enzymes was S-G-G-L-D. The motif was conserved in both argininosuccinate synthase (EC 6.3.4.5) and asparagine synthase (glutamine-hydrolysing) (EC 6.3.5.4). This result showed that the database was useful for the analysis of the relationship between chemical structures and amino acid sequence motifs.