Proposal for a Reaction Mechanism of Hydantoin Racemase Enzymes

Proposal for a Reaction Mechanism of Hydantoin Racemase Enzymes 

The binding affinities of SmeHyuA C76A and C181A active site mutants (alanine mutants) were evaluated for further insight into the role of each residue. Binding of the above substrates to both mutants was also measured by isothermal titration calorimetry which showed that the presence of a proton at the 5-position of the substrate and not in the lateral chain is the critical factor for proper binding. 

Additional binding experiments conducted by fluorescence measurements with C76A mutant and D- and L-isopropylhydantoin and L-ethylhydantoin (D-ethylhydantoin is an inhibitor) showed that this mutant is unable to bind the D-isomers of the substrates. The same experiments carried out with C181A mutant proved that this mutant was not able to bind the L-isomers. These results indicate that cysteine 76 is responsible for the recognition of D-isomers of the 5-monosubstituted hydantoins, whereas cysteine 181 recognizes the L-isomers. 

Once several hydantoin racemases had been studied and characterized, their behavior, together with the other enzymes involved in the hydantoinase process. 

In order to overcome these two problems our group designed a biocatalyst for the production of optically pure D-amino acids from D,L-5-monosubstituted hydan-toins by co-expressing the three genes in one plasmid in a polycistronic structure 

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