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Mutational analysis of the hydantoin hydrolysis pathway in Pseudomonas putida RU-KM3S

  • Applied Genetics and Molecular Biotechnology
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Abstract

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 β-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 β-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 β-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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Acknowledgements

This research was funded by Innovation Fund Grant No. 141141; and G.F.M. was supported by a Rhodes University Henderson Scholarship. The authors wish to acknowledge J.J. Dennis and G. Zylstra for kindly supplying the plasposon vectors, C.J. Hartley, S.A. Clark, and C. Louw for technical assistance, and members of the Rhodes Hydantoinase Research Group for stimulating discussion and constructive advice. The research described in this study was conducted in compliance with the Genetically Modified Organisms Act 15 of 1997[/Sapl4] of South Africa.

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Authors and Affiliations

  1. Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, P.O. Box 94, Grahamstown, 6140, South Africa

    G. F. Matcher & R. A. Dorrington

  2. Department of Chemical Engineering, University of Cape Town, Rondebosch, Cape Town, 7700, South Africa

    S. G. Burton

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  1. G. F. Matcher
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  2. S. G. Burton
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  3. R. A. Dorrington
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Correspondence to R. A. Dorrington.

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Matcher, G.F., Burton, S.G. & Dorrington, R.A. Mutational analysis of the hydantoin hydrolysis pathway in Pseudomonas putida RU-KM3S . Appl Microbiol Biotechnol 65, 391–400 (2004). https://doi.org/10.1007/s00253-004-1597-3

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  • DOI: https://doi.org/10.1007/s00253-004-1597-3

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