Rational Engineering of the Substrate Specificity of a Thermostable D-Hydantoinase (Dihydropyrimidinase).

Hovsep Aganyants,Michèle Lecocq,Haykanush Koloyan,Vehary Sakanyan,Jean-Noël Hallet,Anichka Hovsepyan,Pierre Weigel,Yeranuhi Hovhannisyan,Artur Hambardzumyan

doi:10.3390/ht9010005

Abstract

D-hydantoinases catalyze an enantioselective opening of 5- and 6-membered cyclic structures and therefore can be used for the production of optically pure precursors for biomedical applications. The thermostable D-hydantoinase from Geobacillus stearothermophilus ATCC 31783 is a manganese-dependent enzyme and exhibits low activity towards bulky hydantoin derivatives. Homology modeling with a known 3D structure (PDB code: 1K1D) allowed us to identify the amino acids to be mutated at the substrate binding site and in its immediate vicinity to modulate the substrate specificity. Both single and double substituted mutants were generated by site-directed mutagenesis at appropriate sites located inside and outside of the stereochemistry gate loops (SGL) involved in the substrate binding. Substrate specificity and kinetic constant data demonstrate that the replacement of Phe159 and Trp287 with alanine leads to an increase in the enzyme activity towards D,L-5-benzyl and D,L-5-indolylmethyl hydantoins. The length of the side chain and the hydrophobicity of substrates are essential parameters to consider when designing the substrate binding pocket for bulky hydantoins. Our data highlight that D-hydantoinase is the authentic dihydropyrimidinase involved in the pyrimidine reductive catabolic pathway in moderate thermophiles.

Highlights

Functional, structural and evolutionary relationships of enzymes that cleave the amide ring in cyclic diamide structures have led to the identification of protein families, such as hydrolases, oxidoreductases and amino acid aminotransferases, within a superfamily of amidohydrolytic enzymes [1,2,3]
A significant portion of a 52 kDa hydantoinase of G. stearothermophilus ATCC 31,783 was detected as an aggregated protein in E. coli BL21(DE3)/pET-Hyd cells
Replacements of amino acid residues involved in the metal binding with alanine abolish D-hydantoinase activity [41], and the pH profiles of a metal reconstituted enzyme depend on a particular metal ion bound to the substrate binding site [42]

Summary

Introduction

Functional, structural and evolutionary relationships of enzymes that cleave the amide ring in cyclic diamide structures have led to the identification of protein families, such as hydrolases, oxidoreductases and amino acid aminotransferases, within a superfamily of amidohydrolytic enzymes [1,2,3]. Pure amino acids can be obtained by enantioselective ring opening in 5- and 6-membered hydantoins using cyclic amidohydrolases, known as hydantoinases [1]. It should be recalled that hydantoinase got its conventional name because hydantoin was first obtained by the hydrogenation of allantoin [4]. D-amino acids are naturally tolerant to proteolytic degradation, and hydantoin racemates can be used as promising substrates for the development of semisynthetic antibiotics, peptide hormones, and drugs [5,6,7]. D-specific hydantoinases of the genus Geobacillus have a high level of sequence similarity (Figure S1) that simplifies comparative enzyme assays and the mutual extrapolation of data [10,11,12]. Despite 92% sequence similarity, D-hydantoinase of G. thermocatenulatus has a broader substrate specificity than the enzyme of G. stearothermophilus SD1 [13]

Methods

Results

Discussion

Conclusion