The stereoselectivity and hydrolysis efficiency of recombinant D-hydantoinase from Vigna angularis Against 5-benzylhydantoin derivatives with halogen and methyl substituents.

Gniewomir Latacz,Katarzyna Kieć-Kononowicz

doi:10.1007/s12010-014-1313-4

Gniewomir Latacz, Katarzyna Kieć-Kononowicz

Open Access

https://doi.org/10.1007/s12010-014-1313-4

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Abstract

The researches on d-hydantoinase activity and substrate specificity towards dihydropyrimidine and hydantoin derivatives have been carried out intensively over the last few decades. So far, the major efforts have focused on (R,S)-5-phenylhydantoin and (R,S)-5-(4-hydroxyphenyl)hydantoin, the most desirable d-hydantoinase substrates from pharmaceutical industry point of view. However, it was shown that d-hydantoinase is a substrate-dependent enzyme, and its activity and stereoselectivity towards 5-monosubstituted hydantoins varied significantly with the type of substrate and the source of the enzyme. The aim of this study was to estimate the substrate specificity of d-hydantoinase towards series of 5-benzylhydantoin derivatives with halogen and methyl substituents in the phenyl ring. The biotransformations were carried out by using commercial enzyme: immobilized, recombinant, cloned, and expressed in Escherichia coli d-hydantoinase from Vigna angularis (rD-HYD). All reactions were monitored by capillary electrophoresis (CE), and the conversion yields were calculated. Additionally, enantiomeric ratios of the obtained d-phenylalanine derivatives were estimated by chiral high-performance liquid chromatography (HPLC). Interestingly, the differences in the activities of examined enzyme towards particular 5-benzylhydantoin derivatives were observed. CE was also shown as a promising method for monitoring the hydrolysis of new substrates by d-hydantoinase and further analyzing of enzyme substrate specificity.

Highlights

Dihidropyrimidinase (EC 3.5.2.2) is a very important enzyme belonging to the cyclic amidohydrolases superfamily which take part in either pyrimidines or purines catabolism in bacteria, yeasts, plants, and animals
The hydantoinase process has been successfully used for years in pharmaceutical industry for biosynthesis of D-phenylglycine and D-4-hydroxyphenylglicyne starting from (R,S)-5phenylhydantoin and (R,S)-4-hydroxyphenylhydantoin [5]
From a pharmaceutical point of view, D-amino acids are important as the chiral building blocks of new peptidomimetics

Summary

Introduction

Dihidropyrimidinase (EC 3.5.2.2) is a very important enzyme belonging to the cyclic amidohydrolases superfamily which take part in either pyrimidines or purines catabolism in bacteria, yeasts, plants, and animals. This enzyme, known as hydantoinase, is commonly used in hydantoinase method for obtaining optically pure D- or L-amino acids starting from racemic 5-monosubstituted hydantoins. One of the preferred D-amino acids to design and synthesis of new peptidomimetics is D-phenylalanine (D-phe). D-phe was used for the synthesis of the analgesic opioid peptide Ac-rfwink-NH2 [11], and nateglinide— drug stimulating the release of insulin [12] Another important peptidomimetics containing Dphe are the analogs of somatostatin hormone, such as synthetic somatostatin analogs: octreotide and vapreotide [13, 14] or heksarelin, a somatostatin-releasing stimulator [15]. In view of increasing importance of D-phe in the design of new peptidomimetics, the hydantoinase method was successfully applied for the synthesis of the series of halogen and methyl D-phe derivatives. Taking into account the substrate-dependent properties of Dhydantoinases, the D-hydantoinase from Vigna angularis—immobilized, recombinant, and expressed in Escherichia coli—was used to estimate its substrate specificity towards abovementioned 5-benzylhydantoin derivatives

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