Characterization of the hydantoin- hydrolysing system of Pseudomonas putida RU-KM3S Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy of Rhodes University by Gwynneth Felicity Matcher April 2004 ABSTRACT The biocatalytic conversion of 5-monosubstituted hydantoin derivatives to optically pure amino acids involves two reaction steps: the hydrolysis of hydantoin to N-carbamylamino acid by an hydantoinase or dihydropyrimidinase enzyme, followed by conversion of the N- carbamylamino acid to the corresponding amino acid by an N-carbamoylase enzyme. This biocatalytic process has been successfully applied in several industrial processes for the production of enantiomerically pure amino acids used in the synthesis of pharmaceuticals, insecticides, hormones, and food additives. P. putida RU-KM3S was selected for study based on inherent high levels of hydantoinase and N-carbamoylase activity. Subsequent biocatalytic analysis of the enzyme activity within this strain revealed unique properties thus prompting further characterization. The main focus of this research was the isolation of the genes encoding the hydantoin-hydrolysing pathway in RU-KM3S. A genomic library was constructed and screened for heterologous expression of the hydantoin-hydrolysing enzymes. However, this approach was unsuccessful prompting the use of transposon mutagenesis in order to circumvent the drawbacks associated with complementation studies. The enzymes responsible for hydantoin-hydrolysis were identified by insertional inactivation as a dihydropyrimidinase and β-ureidopropionase encoded by dhp and bup respectively. A third open reading frame, encoding a putative transport protein, was identified between the dhp and bup genes and appeared to share a promoter with bup. Analysis of the amino acid sequence deduced from bup and dhp substantiated the distinctive properties and potential industrial application of the L-enantioselective β-ureidopropionase and provided targets for potential optimisation of the substrate-selectivity and activity of the dihydropyrimidinase by site directed mutagenesis. Several transposon-generated mutants with an altered phenotype for growth on minimal medium with hydantoin as the sole source of nitrogen were also isolated. Analysis of the insertion events in these mutants revealed disruptions of genes encoding key elements of the Ntr global regulatory pathway. However, inactivation of these genes had no effect on the dihydropyrimidinase and β-ureidopropionase activity levels. An additional mutant in which the gene coding for the dihydrolipoamide succinyltransferase, which is involved in the TCA cycle, was isolated with reduced levels of both dihydropyrimidinase and β-ureidopropionase activities. These results indicated that the hydantoin-hydrolysis pathway in RU-KM3S is regulated by carbon rather than nitrogen catabolite repression. This was confirmed by the reduction of hydantoin-hydrolysis in cells grown in excess carbon as opposed to nitrogen. Identification of a putative CRP-binding site within the promoter region of these enzymes further supported the regulatory role of carbon catabolite repression (CCR). As CCR in Pseudomonads is poorly understood, elucidation of the mechanism by which the hydantoin- hydrolysing pathway in RU-KM3S is regulated would provide valuable insight into this complex process. I TABLE OF CONTENTS List of Figures......................................................................................................................... II List of Tables V List of Abbreviations VII Acknowledgements............................................................................................................... VIII Research outputs IX Chapter 1: Literature review................................................................................... 1 Chapter 2: Identification and biocatalytic characterization of strain RU-KM3S 45 Chapter 3: Construction and screening of RU-KM3S genome library................... 64 Chapter 4: Mutagenesis of RU-KM3S .......................................................................................................... 89 Chapter 5: Structural and regulatory components of the hydantoin-hydrolysing pathway of RU-KM3S ...................................................................................................................... 107 Chapter 6: Analysis of the bup and dhp genes from RU-KM3S 127 Chapter 7: General conclusions 149 Appendices................................................................................................................ 158 References 174 II LIST OF FIGURES Figure 1.1 Enzymatic hydrolysis of hydantoin to form enantiomerically pure amino acids 5 Figure 1.2 Keto-enol-tautomerism of 5-monosubstituted hydantoins.......................................... 24 Figure 1.3 Ribbon representation of the Thermus sp. D-hydantoinase quaternary structure (A), monomer (B), and catalytic site (C)............................................................................. 26 Figure 1.4 Hydrogen bond formation between the D-hydantoinase and D-5-monosubstituted hydantoin substrate (A), and the steric clash occurring when an L-5-monosubstitued hydantoin substrate is modelled into this site (B)........................................................ 27 Figure 1.5 Ribbon representation of the D-carbamoylase quaternary structure (A), monomer (B), and catalytic site (C) ....................................................................................................30 Figure 1.6 Model of the catalytic binding pocket of the D-carbamoylase with hydroxyphenyl- hydantoin substrate (A) and the interactions of the substrate with the enzyme (B) 30 Figure 2.1 Rational development of a biocatalyst for production of industrially important organic compounds ....................................................................................................................45 Figure 2.2 Production of N-carbamylglycine and glycine from hydantoin by RU-KM3S at various phases of growth in complete medium......................................................................... 50 Figure 2.3 The effect of divalent cations on hydantoinase and N-carbamoylase activity after dialysis of cell free extracts of RU-KM3S against phosphate buffer containing Na2EDTA .......................................................................................................................54 Figure 2.4 Effect of ATP on hydantoinase and N-carbamoylase activities in resting whole cell biocatalytic assays of RU-KM3S............................................................................................................................ 55 Figure 2.5 DNA fragments generated by restriction endonuclease digestion of the 16S rRNA gene of RU-KM3S and separated electophoretically through an agarose gel 56 Figure 2.6 Restriction endonuclease map of the 16S rRNA gene from P. putida RU-KM3S constructed from sequence data obtained from the parental and deletion constructs of the PCR product in pUC18 57 Figure 2.7 Boxshade of the aligned 16S rRNA gene sequences of P. putida strains BH and RU-KM3S ...................................................................................................................................................................................60 Figure 3.1 Electrophoresis of the native plasmid extracted from RU-KM3S as well as RU-KM3S chromosomal DNA (A) and plasmid extraction supernatant for RU-KM3S and GMPpc (B) 71 Figure 3.2 Partial digestion of RU-KM3S chromosomal DNA by addition of varying concentrations of Sau 3A1..................................................................................................................... 72 Figure 3.3 Estimation of insertion frequency and average size of insert of the recombinant clones in the constructed genomic library of RU-KM3S....................................................................................... 73 Figure 3.4 Schematic diagram of the pBK-CMV vector in which the genomic library of RU-KM3S was constructed............................................................................................................. 76 Figure 3.5 Endonuclease digestion of plasmid preparations (A) probed with radioactive PCR product of the insert from pGMLib15 (B) 77 III Figure 3.6 Southern analysis of the chromosomal DNA flanking the sequence encoded by the insert in pGMLib15........................................................................................................ 79 Figure 3.7 Graphical representation of the strategy used to determine the nucleotide sequence of the RU-KM3S genomic fragment contained in pGMlib15........................................... 81 Figure 3.8 Restriction endonuclease digestion of pGMlib1 and pGMlib2 from initial culture of the recombinant directly from the NCG-MM plate (A), and subsequent to sub-culturing in Luria broth (B) 83 Figure 3.9 Comparison of the growth of E. coli DH5α transformants expressing pBK-CMV, pGMlib1, or pGMlib2 over time.................................................................................... 84 Figure 4.1 Schematic map of the plasposon pTnMod-OKm ........................................................90 Figure 4.2 Optimisation of EMS mutagenesis treatment of RU-KM3S cells................................ 94 Figure 4.3 Phenotypes of RU-KM3S wild type (WT) and corresponding chemically induced mutant strains GMP1 and GMP2 on nutrient agar plates supplemented with and without 0.005% 5-FU, in the presence and absence of hydantoin as