Molecular Cloning and Expression of thehyuGenes fromMicrobacterium liquefaciensAJ 3912, Responsible for the Conversion of 5-Substituted Hydantoins to α-Amino Acids, inEscherichia coli

Abstract

A DNA fragment from Microbacterium liquefaciens AJ 3912, containing the genes responsible for the conversion of 5-substituted-hydantoins to -amino acids, was cloned in Escherichia coli and sequenced. Seven open reading frames (hyuP, hyuA, hyuH, hyuC, ORF1, ORF2, and ORF3) were identified on the 7.5 kb fragment. The deduced amino acid sequence encoded by the hyuA gene included the N-terminal amino acid sequence of the hydantoin racemase from M. liquefaciens AJ 3912. The hyuA, hyuH, and hyuC genes were heterologously expressed in E. coli; their presence corresponded with the detection of hydantoin racemase, hydantoinase, and N-carbamoyl -amino acid amido hydrolase enzymatic activities respectively. The deduced amino acid sequences of hyuP were similar to those of the allantoin (5-ureido-hydantoin) permease from Saccharomyces cerevisiae, suggesting that hyuP protein might function as a hydantoin transporter.

The Hydantoin Transport Protein from Microbacterium liquefaciens

“…This outcome illustrates the power of comparative genomics to infer functions of genes, in this case, from a combination of both the prediction that an encoded protein is a transporter by its clustering by long-range homology in the NCS-1 family and also the occurrence of its gene in an operon encoding putatively related metabolic enzymes. The preferred substrates were found to be L-5-indolylmethyl-hydantoin and L-5-benzyl-hydantoin, consistent with the chiral specificity for the L form of both the associated hydantoinase and N-carbamoylase in M. liquefaciens AJ3912 (24,36). Thus, the transport protein is deduced to play a role in the metabolism of tryptophan, phenylalanine, and probably tyrosine.…”

“…The hyu genes encoding the enzymes often form gene clusters, indicating the existence of coherent roles for the combination of hydantoinase, N-carbamoylase, and HRase (6,24,29,30,33). Among these genes for the three hydantoin metabolic enzymes, we found genes encoding putative transporters, for example in the hyu gene clusters from Pseudomonas sp.…”

“…Among these genes for the three hydantoin metabolic enzymes, we found genes encoding putative transporters, for example in the hyu gene clusters from Pseudomonas sp. NS671 (29,30), Arthrobacter aurescens DSM 3747 (33), and Microbacterium liquefaciens AJ 3912 (24) (Fig. 1).…”

Evaluation of substrate promiscuity of anL‐carbamoyl amino acid amidohydrolase fromGeobacillus stearothermophilusCECT43

“…5 The chirality of the amino acid obtained depends on the stereospecificity of the last enzyme in the reaction cascade (N-carbamoyl-L-amino-acid amidohydrolases, also known as L-Ncarbamoylases). 6 This enzyme has been found in several microorganisms of the genera Arthrobacter, 7 Alcaligenes, 8 Bacillus, 9 Blastobacter, 10 Flavobacterium, 11 Microbacterium, 12 Pseudomonas, 13 and Sinorhizobium.…”

Crystallization and preliminary crystallographic studies of an active-site mutant hydantoin racemase fromSinorhizobium melilotiCECT4114

“…Some hydantoin racemase-encoding genes have been found together with a carbamoylase and a hydantoinase/ dihydropyrimidinase including a potential hydantoin transporter enzyme (Hils et al, 2001;Suzuki, Takenaka et al, 2005;Watabe et al, 1992b;Wiese et al, 2001); this activity has recently been suggested for the corresponding gene from Microbacterium liquefaciens (Suzuki & Henderson, 2006). However, the most common application of these enzymes is the use of the 'hydantoinase process', a cheap and environmentally friendly enzymatic method for the potential production of any optically pure natural or unnatural amino acid from a wide spectrum of d,l-5-monosubstituted hydantoins Martinez-Rodriguez et al, 2002).…”