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Title: Physical and functional analysis of genes from the CAM catabolic plasmid encoding probable steps in the catabolism of camphor
Author: Tongyoo, Narongchai
ISNI:       0000 0001 3535 0615
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2002
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The ability to grow on either (+) or (-)-camphor, a bicyclic monoterpene ketone, of Pseudomonas putida NCIMB 10007 is conferred by the CAM plasmid, which has catabolic genes encoding enzymes that specify camphor degradation. Cloning of camR (cam repressor gene), camD (5-exo-hydroxycamphor dehydrogenase gene), camC (cytochrome P450cam gene), camA (putidaredoxin reductase gene) and camB (putidaredoxin gene) which are responsible for early steps of camphor degradation have been previously described. However, a study of the CAM plasmid involved in the further steps in camphor degradation has not been carried out. In this study, the nucleotide sequence of the 4485 bp on the left-hand side region of the cam operon on the CAM plasmid has been cloned. This nucleotide sequence consists of four possible open reading frames; orf1, orf2, orf3 and orf4. The deduced amino acid of orf1 shares identity with steroid monooxygenase (Rhodococcus rhodochrous), cyclohexanone monooxygenase (Brevibacterium sp. HCU) and cyclohexanone 1,2-monooxygenase (Acinetobacter sp. NCIMB 9871). Yet, the nucleotide sequence of orf1 is incomplete and is probably missing its 5'end sequence. The deduced amino acid sequence of orf2 is similar to putative limonene monooxygenase (Rhodococcus erythropolis), alkanal monooxygenase (Xenorhabdus luminescens HW) and luciferase related proteins. Moreover, the N-terminus of orf2 deduced amino aicd is homologous (80% identity) with that of 2,5-diketocamphane 1,2-monooxygenase, an enzyme in the third step of camphor degradation. The Orf3 protein shares identity and similarity with a number of transcriptional regulator proteins in the tetR family. The deduced amino acid sequence of orf4 is highly related to the methyl parathion hydrolase (Plesiomonas sp M6), methyl parathion degrading protein (Plesiomonas sp. DLL-1), and other proteins in the beta-metallo-lactamase family. A 40.7 kDa Orf2 monooxygenase was purified to homogeneity. However, the Orf2 monooxygenase has no activity towards (+)-and (-)- limonene, (+)-and (-)-pinene and cyclohexanone in the reaction with NADPH or NADH. The Orf4 protein is a membrane associated hydrolase possessing 29-leader signal peptide at its N-terminus. This signal peptide is; however, unprocessed by signal peptidease in Escherichia coli. Although whole cell activities using E. coli BL21(DE3)CodonPlus-RP harbouring pQR424 showed that Orf4 hydrolase is capable of hydrolysing [gamma]-butyrolactone and paraoxon with high initial hydrolysis rates of 84.2 and 13.0 mol/h/mg-dry-cell weight respectively, the natural substrate for Orf4 hydrolase remains unknown.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available