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

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.

Reply to the Comment by Littlechild and Isupov

“…Firstly, all the observations made in doi:10.3390/microorganisms4040038 [ 1 ] about the “model/structural model” refer exclusively to material presented in Sections 3.5–3.7 in [ 2 ]—none of the comments refer to the experimentally determined crystalline structures of 3,6-DKCMO (PDB code: 4UMW) presented in Sections 3.2 and 3.3. This caveat is also relevant to observations made elsewhere [ 3 , 4 ].…”

“…Secondly, Drs. Littlechild and Isupov’s assertion that “In the paper [ 1 ] the lead author Willetts has described work carried out by himself and Dr David Kelly prior to 2008” is incorrect. As evidenced below ( vide supra ), correct facts and timelines are both important matters: therefore, to reiterate the statement made in the “Author Contribution” entry of [ 1 ], while Dr. David Kelly’s major contribution was to the conception and overall design of the research programme prior to his sudden unexpected death in 2008, most of the subsequent stages (gathering and analysing data, and writing the paper) were conducted exclusively thereafter by Andrew Willetts.…”

“…Important to gaining a full comprehension of both the issues raised by Drs. Littlechild and Isupov’s Comment and the prior observations made [ 1 , 3 , 4 ] regarding Isupov et al’s modelling studies is the fact that the oxidation of reduced flavins by oxygen-dependent enzymes, including Type I and II Baeyer-Villiger monooxygenases (BVMOs: [ 6 ]), has been extensively studied for several decades. Consequently, both structural and functional aspects of a significant number of representative examples, including various flavin-diffusible two-component monooxygenases (fd-TCMOs: [ 7 ]), are very well characterised and understood.…”

“…There are three principal important biochemical issues that are likely to be influential: In terms of function, the difference between a flavoprotein-type monooxygenase and a flavin-diffusible monooxygenase ( vide infra ) is crucial. In the specific case of 3,6-DKCMO, this is reflected starkly by the fact that relevant kinetics confirm that the respective Kd values for FMN and FNR differ by >500% [ 1 ]. This indicates that while the reduced form of the flavin is bound very tightly in the active site of the enzyme, FMN exhibits poor affinity (as commented on a number of times by Isupov et al [ 2 ]), implying that any relevant bonding will be weak, and probably largely random, as recorded with a number of other flavin-diffusible TCMOs [ 7 ].…”

Comments to Article by Willetts A. et al., Microorganisms 2016, 4, 38

“…We would like to comment on recent work published in your journal in October 2016 by Willetts A. et al [ 1 ]. In the paper, the lead author Willetts describes work carried out by himself and Dr. David Kelly prior to 2008.…”