Mutational analysis of the hydantoin hydrolysis pathway in Pseudomonas putida RU-KM3S.
The biocatalytic conversion of 5-mono-substituted hydantoins to the corresponding D-amino acids or L-amino acids involves first the hydrolysis of hydantoin to a N-carbamoylamino acid by an hydantoinase or dihydropyrimidinase, followed by the conversion of the N-carbamoylamino acid to the amino acid by N-carbamylamino acid amidohydrolase ( N-carbamoylase). Pseudomonas putida strain RU-KM3S, with high levels of hydantoin-hydrolysing activity, has been shown to exhibit non-stereoselective hydantoinase and L-selective N-carbamoylase activity. This study focused on identifying the hydantoinase and N-carbamoylase-encoding genes in this strain, using transposon mutagenesis and selection for altered growth phenotypes on minimal medium with hydantoin as a nitrogen source. Insertional inactivation of two genes, dhp and bup, encoding a dihydropyrimidinase and beta-ureidopropionase, respectively, resulted in loss of hydantoinase and N-carbamoylase activity, indicating that these gene products were responsible for hydantoin hydrolysis in this strain. dhp and bup are linked to an open reading frame encoding a putative transport protein, which probably shares a promoter with bup. Two mutant strains were isolated with increased levels of dihydropyrimidinase but not beta-ureidopropionase activity. Transposon mutants in which key elements of the nitrogen regulatory pathway were inactivated were unable to utilize hydantoin or uracil as a nitrogen source. However, these mutations had no effect on either the dihydropyrimidinase or beta-ureidopropionase activity. Disruption of the gene encoding dihydrolipoamide succinyltransferase resulted in a significant reduction in the activity of both enzymes, suggesting a role for carbon catabolite repression in the regulation of hydantoin hydrolysis in P. putida RU-KM3S cells.
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Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, PO Box 94, 6140, Grahamstown, South Africa.
Burton
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Dorrington
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RA
D000217:Acyltransferases / Q000378:metabolism; D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology; D003470:Culture Media / Q000737:chemistry; D005798:Genes, Bacterial; D006827:Hydantoins / Q000378:metabolism*; D006868:Hydrolysis; D016254:Mutagenesis, Insertional; D009154:Mutation; D009584:Nitrogen / Q000378:metabolism; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D014498:Uracil / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003470:Culture Media; D006827:Hydantoins; D014498:Uracil; D000217:Acyltransferases; C021515:dihydrolipoamide succinyltransferase; D000581:Amidohydrolases; C451299:N-carbamoylamino acid amidohydrolase; C021590:beta-ureidopropionase; C020047:dihydropyrimidinase; D009584:Nitrogen