Functional expression and characterization of the two cyclic amidohydrolase enzymes, allantoinase and a novel phenylhydantoinase, from Escherichia coli

A superfamily of cyclic amidohydrolases, including dihydropyrimidinase, all antoinase, hydantoinase, and dihydroorotase, all of which are involved in t he metabolism of purine and pyrimidine rings, was recently proposed based o n the rigidly conserved structural domains in identical positions of the re lated enzymes. With these conserved domains, two putative cyclic amidohydro lase genes from Escherichia coli, flanked by related genes, were identified and characterized. From the genome sequence of E. coli, the allB gene and a putative open reading frame, tentatively designated as a hyuA (for hydant oin-utilizing enzyme) gene, were predicted to express hydrolases, In contra st to allB, high-level expression of hyuA in E. coli of a single protein wa s unsuccessful even under various induction conditions. We expressed HyuA a s a maltose binding protein fusion protein and AllB in its native form and then purified each of them by conventional procedures. allB was found to en code a tetrameric allantoinase (453 amino acids) which specifically hydroly zes the purine metabolite allantoin to allantoic acid. Another open reading frame, hyuA, located near 64.4 min on the physical map and known as a UUG start, coded for D-stereospecific phenylhydantoinase (465 amino acids) whic h is a homotetramer. As a novel enzyme belonging to a cyclic amidohydrolase superfamily, E. coli phenylhydantoinase exhibited a distinct activity towa rd the hydantoin derivative with an aromatic side chain at the 5' position but did not readily hydrolyze the simple cyclic ureides. The deduced amino acid sequence of the novel phenylhydantoinase shared a significant homology (>45%) with those of allantoinase and dihydropyrimidinase, but its functio nal role still remains to be elucidated. Despite the unclear physiological function of HyuA, its presence, along with the allantoin-utilizing AllB, st rongly suggested that the cyclic ureides might be utilized as nutrient sour ces in E. coli.