Flavin-Dependent Redox Transfers by the Two-Component Diketocamphane Monooxygenases of Camphor-Grown Pseudomonas putida NCIMB 10007.

Andrew Willetts,David Kelly

doi:10.3390/microorganisms4040038

Andrew Willetts, David Kelly

Open Access

https://doi.org/10.3390/microorganisms4040038

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Journal: Microorganisms	Publication Date: Oct 13, 2016
Citations: 45	License type: CC BY 4.0

Affiliation: Cardiff University, University of Exeter

Abstract

The progressive titres of key monooxygenases and their requisite native donors of reducing power were used to assess the relative contribution of various camphor plasmid (CAM plasmid)- and chromosome-coded activities to biodegradation of (rac)-camphor at successive stages throughout growth of Pseudomonas putida NCIMB 10007 on the bicylic monoterpenoid. A number of different flavin reductases (FRs) have the potential to supply reduced flavin mononucleotide to both 2,5- and 3,6-diketocamphane monooxygenase, the key isoenzymic two-component monooxygenases that delineate respectively the (+)- and (−)-camphor branches of the convergent degradation pathway. Two different constitutive chromosome-coded ferric reductases able to act as FRs can serve such as role throughout all stages of camphor-dependent growth, whereas Fred, a chromosome-coded inducible FR can only play a potentially significant role in the relatively late stages. Putidaredoxin reductase, an inducible CAM plasmid-coded flavoprotein that serves an established role as a redox intermediate for plasmid-coded cytochrome P450 monooxygenase also has the potential to serve as an important FR for both diketocamphane monooxygenases (DKCMOs) throughout most stages of camphor-dependent growth.

Highlights

The bicyclic monoterpenoid camphor, present as either separate enantiomer or a racemic mixture, is a molecule that is encountered relatively rarely in the biosphere [1]
Gunsalus et al [6] established that when P. putida NCIMB 10007 was inoculated into a defined medium containing both succinate and-camphor-U-C14, diauxic growth was observed whereby all available succinate was completely utilised during the first growth phase, with no CO2 derived from camphor released before commencement of the subsequent phase of exclusively camphor-dependent growth
By extending these prior studies to monitor the specific activities of relevant enzymes in cell-free extracts prepared at timed intervals throughout these successive stages of growth, it should be possible to establish the importance of potentially key enzymes involved in FNR generation and deployment

Summary

Introduction

The bicyclic monoterpenoid camphor, present as either separate enantiomer or a racemic mixture, is a molecule that is encountered relatively rarely in the biosphere [1]. It was for this reason that camphor was chosen to initiate a series of studies on microbial biodegradation as a model of how an “atypical” molecule can become integrated into the TCA cycle and other shared central pathways of metabolism [2,3]. The relevant plasmid-coded homodimeric oxygenating moieties for both DKCMOs receive unbound reduced flavin mononucleotide (FNR) by rapid free diffusion, not as previously believed from a plasmid-coded oxidase/dehydrogenase [10,11], but from Fred, a chromosome-coded enzyme [5]. The extremely high purity (>99.9% ee) of the chiral products of biotransfomations catalysed by a mixture of the two 2,5-DKCMO isoenzymes [8,13,14] strongly suggests that both enzymes have very similar catalytic specificities

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