The Role of Dioxygen in Microbial Bio-Oxygenation: Challenging Biochemistry, Illustrated by a Short History of a Long Misunderstood Enzyme.

A Special Issue of Microorganisms devoted to 'Microbial Biocatalysis and Biodegradation' would be incomplete without some form of acknowledgement of the many important roles that dioxygen-dependent enzymes (principally mono- and dioxygenases) play in relevant aspects of bio-oxygenation. This is reflected by the multiple strategic roles that dioxygen -dependent microbial enzymes play both in generating valuable synthons for chemoenzymatic synthesis and in facilitating reactions that help to drive the global geochemical carbon cycle. A useful insight into this can be gained by reviewing the evolution of the current status of 2,5-diketocamphane 1,2-monooxygenase (EC 1.14.14.108) from (+)-camphor-grown Pseudomonas putida ATCC 17453, the key enzyme that promotes the initial ring cleavage of this natural bicyclic terpene. Over the last sixty years, the perceived nature of this monooxygenase has transmogrified significantly. Commencing in the 1960s, extensive initial studies consistently reported that the enzyme was a monomeric true flavoprotein dependent on both FMNH2 and nonheme iron as bound cofactors. However, over the last decade, all those criteria have changed absolutely, and the enzyme is currently acknowledged to be a metal ion-independent homodimeric flavin-dependent two-component mono-oxygenase deploying FMNH2 as a cosubstrate. That transition is a paradigm of the ever evolving nature of scientific knowledge.

Bicyclo[3.2.0]carbocyclic Molecules and Redox Biotransformations: The Evolution of Closed-Loop Artificial Linear Biocatalytic Cascades and Related Redox-Neutral Systems.

The role of cofactor recycling in determining the efficiency of artificial biocatalytic cascades has become paramount in recent years. Closed-loop cofactor recycling, which initially emerged in the 1990s, has made a valuable contribution to the development of this aspect of biotechnology. However, the evolution of redox-neutral closed-loop cofactor recycling has a longer history that has been integrally linked to the enzymology of oxy-functionalised bicyclo[3.2.0]carbocyclic molecule metabolism throughout. This review traces that relevant history from the mid-1960s to current times.

Inter-Species Redox Coupling by Flavin Reductases and FMN-Dependent Two-Component Monooxygenases Undertaking Nucleophilic Baeyer-Villiger Biooxygenations.

Using highly purified enzyme preparations throughout, initial kinetic studies demonstrated that the isoenzymic 2,5- and 3,6-diketocamphane mono-oxygenases from Pseudomonas putida ATCC 17453 and the LuxAB luciferase from Vibrio fischeri ATCC 7744 exhibit commonality in being FMN-dependent two-component monooxygenases that promote redox coupling by the transfer of flavin reductase-generated FMNH2 by rapid free diffusion. Subsequent studies confirmed the comprehensive inter-species compatibility of both native and non-native flavin reductases with each of the tested monooxygenases. For all three monooxygenases, non-native flavin reductases from Escherichia coli ATCC 11105 and Aminobacter aminovorans ATCC 29600 were confirmed to be more efficient donators of FMNH2 than the corresponding tested native flavin reductases. Some potential practical implications of these outcomes are considered for optimising FMNH2-dependent biooxygenations of recognised practical and commercial value.

The Isoenzymic Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453-An Episodic History and Still Mysterious after 60 Years.

Researching the involvement of molecular oxygen in the degradation of the naturally occurring bicyclic terpene camphor has generated a six-decade history of fascinating monooxygenase biochemistry. While an extensive bibliography exists reporting the many varied studies on camphor 5-monooxygenase, the initiating enzyme of the relevant catabolic pathway in Pseudomonas putida ATCC 17453, the equivalent recorded history of the isoenzymic diketocamphane monooxygenases, the enzymes that facilitate the initial ring cleavage of the bicyclic terpene, is both less extensive and more enigmatic. First referred to as 'ketolactonase-an enzyme for cyclic lactonization'-the enzyme now classified as 2,5-diketocamphane 1,2-monooxygenase (EC 1.14.14.108) holds a special place in the history of oxygen-dependent biochemistry, being the first biocatalyst confirmed to undertake a biooxygenation reaction equivalent to the peracid-catalysed Baeyer-Villiger chemical oxidation first reported in the late 19th century. However, following that auspicious beginning, the biochemistry of EC 1.14.14.108, and its isoenzymic partner 3,6-diketocamphane 1,6-monooxygenase (EC 1.14.14.155) was dogged for many years by the mistaken belief that the enzymes were true flavoproteins that function with a tightly-bound flavin cofactor in the active site. This misconception led to a number of erroneous interpretations of relevant experimental data. It is only in the last decade, initially as the result of pure serendipity, that these enzymes have been confirmed to be members of a relatively recently discovered class of oxygen-dependent enzymes, the flavin-dependent two-component monooxygenases. This has promoted a renaissance of interest in the enzymes, resulting in programmes of research that have significantly expanded current knowledge of both their mode of action and regulation in camphor-grown P. putida ATCC 17453. However, some features of the biochemistry of the isoenzymic diketocamphane monooxygenases remain currently unexplained. It is the episodic history of these enzymes and some of what remains unresolved that are the principal subjects of this review.

Conferring the Metabolic Self-Sufficiency of the CAM Plasmid of Pseudomonas putida ATCC 17453: The Key Role of Putidaredoxin Reductase.

The relative importance of camphor (CAM) plasmid-coded putidaredoxin reductase (PdR) and the chromosome-coded flavin reductases Frp1, Frp2 and Fred for supplying reduced FMN (FNR) to the enantiocomplementary 2,5- and 3,6-diketocamphane monooxygenases (DKCMOs) that are essential for the growth of Pseudomonas putida ATCC 17453 on (rac)-camphor was examined. By undertaking studies in the time window prior to the induction of Fred, and selectively inhibiting Frp1 and 2 with Zn2+, it was confirmed that PdR could serve as the sole active supplier of FNR to the DKCMOs. This establishes for the first time that the CAM plasmid can function as an autonomous extrachromosomal genetic element able to express all the enzymes and redox factors necessary to ensure entry of the C10 bicyclic terpene into the central pathways of metabolism via isobutyryl-CoA.

Characterised Flavin-Dependent Two-Component Monooxygenases from the CAM Plasmid of Pseudomonas putida ATCC 17453 (NCIMB 10007): ketolactonases by Another Name.

The CAM plasmid-coded isoenzymic diketocamphane monooxygenases induced in Pseudomonas putida ATCC 17453 (NCIMB 10007) by growth of the bacterium on the bicyclic monoterpene (rac)-camphor are notable both for their interesting history, and their strategic importance in chemoenzymatic syntheses. Originally named 'ketolactonase-an enzyme system for cyclic lactonization' because of its characterised mode of action, (+)-camphor-induced 2,5-diketocamphane 1,2-monooxygenase was the first example of a Baeyer-Villiger monooxygenase activity to be confirmed in vitro. Both this enzyme and the enantiocomplementary (-)-camphor-induced 3,6-diketocamphane 1,6-monooxygenase were mistakenly classified and studied as coenzyme-containing flavoproteins for nearly 40 years before being correctly recognised and reinvestigated as FMN-dependent two-component monooxygenases. As has subsequently become evident, both the nature and number of flavin reductases able to supply the requisite reduced flavin co-substrate for the monooxygenases changes progressively throughout the different phases of camphor-dependent growth. Highly purified preparations of the enantiocomplementary monooxygenases have been exploited successfully for undertaking both nucleophilic and electrophilic biooxidations generating various enantiopure lactones and sulfoxides of value as chiral synthons and auxiliaries, respectively. In this review the chequered history, current functional understanding, and scope and value as biocatalysts of the diketocamphane monooxygenases are discussed.

Regulation of Camphor Metabolism: Induction and Repression of Relevant Monooxygenases in Pseudomonas putida NCIMB 10007.

For the first time, the differential rates of synthesis of all the key monooxygenases involved in the catabolism by Pseudomonas putida NCIMB 10007 of bicyclic (rac)-camphor to ∆2,5-3,4,4-trimethylpimelyl-CoA, the first aliphatic pathway intermediate, have been determined to help establish the relevant induction profile of each of the oxygen-dependent enzymes. The efficacy of both relevant substrates and pathway metabolites as inducers has been established. Further, inhibitors with characterised functionality have been used to indicate that the pertinent regulatory controls operate at the level of transcription of the corresponding genes.

Modelling water diffusion in plasticizers: development and optimization of a force field for 2,4-dinitroethylbenzene and 2,4,6-trinitroethylbenzene.

A classical all-atom force field has been developed for 2,4,6-trinitroethylbenzene and 2,4-dinitroethylbenzene and applied in molecular dynamics simulations of the two pure and two mixed plasticizer systems. Bonding parameters and partial charges were derived through electronic and geometry optimization of the single molecules. The other required parameters were derived from values already available in the literature for generic nitro aromatic compounds, which were adjusted to reproduce to a high level of accuracy the densities of 2,4-dinitroethylbenzene, 2,4,6-trinitroethylbenzene and the energetic plasticizers K10 and R8002. This force field has been applied to both K10 and R8002, which when used as plasticizers form an energetic binder with nitrocellulose. Nitrocellulose decomposes in storage, under varying conditions, but in particular where it may become increasingly dry. Following the derivation of the force field, we have therefore applied it to calculate water diffusion coefficients for each of the different materials at 298 K and 338 K, thereby providing a starting point for understanding water behaviour in a nitrocellulose binder.

Reply to the Comment by Littlechild and Isupov.

I thank Drs. Littlechild and Isupov for their recent comments, which are considered below. Before addressing these specifically, their correspondence raises two more general issues which require initial clarification.[...].

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

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.

Multiple native flavin reductases in camphor-metabolizing Pseudomonas putida NCIMB 10007: functional interaction with two-component diketocamphane monooxygenase isoenzymes.

Although they have been studied for nearly 50 years, the source of the FMNH2 needed for effective biooxidation by the 2,5- and 3,6-diketocamphane monooxygenase (DKCMO) isoenzymes induced by the growth of Pseudomonas putida NCIMB 10007 (ATCC 17453) on camphor remains incompletely characterized. Prior studies have focussed exclusively on enzymes present in cells harvested during late-exponential-phase growth despite considerable circumstantial evidence that the flavin reductase (FR) component of these multicomponent monooxygenases is subject to growth-phase-dependent variation. In this study, a number of alternative FMNH2-generating activities, including both conventional FRs and enzymes also able to serve as ferric reductases, were isolated from camphor-grown cells, and the relative level, and hence potential contribution, of these various proteins shown to vary considerably depending on the point of harvest of NCIMB 10007 within exponential-phase growth. While two constitutive monomeric ferric reductases (molecular masses 27.0 and 28.5 kDa) were found to be the major relevant sources of FMNH2 during the initial stages of growth on camphor-based media, a significant subsequent contribution throughout the mid- to late-exponential phases of growth was also made by the camphor-induced homodimeric 37.0 kDa FR Fred, recently reported to serve such a role exclusively. The possible involvement of camphor-induced putidaredoxin reductase (51.0 kDa) as a contributory activity was also investigated and considered. Studies with highly purified preparations of the isofunctional DKCMOs confirmed the potential of the various reductases to function effectively as sources of the requisite FMNH2 to both monooxygenases at different times throughout growth on camphor.

Functional assembly of camphor converting two-component Baeyer-Villiger monooxygenases with a flavin reductase from E. coli.

The major limitation in the synthetic application of two-component Baeyer-Villiger monooxygenases was addressed by identifying the 28-kDa flavin-reductase Fre from Escherichia coli as a suitable supplier of reduced FMN for these enzymes. Coexpression of Fre with either 2,5- or 3,6-diketocamphane monooxygenase from Pseudomonas putida NCIMB 10007 significantly enhanced the conversion of camphor and norcamphor serving as representative ketones. With purified enzymes, full conversion was achieved, while only slight amounts of product were formed in the absence of this flavin reductase. Fusion of the genes of Fre and DKCMOs into single open reading frame constructs resulted in unstable proteins exhibiting flavin reducing, but poor oxygenating activity, which led to overall decreased conversion of camphor.

The biodiscovery potential of marine bacteria: an investigation of phylogeny and function.

A collection of marine bacteria isolated from a temperate coastal zone has been screened in a programme of biodiscovery. A total of 34 enzymes with biotechnological potential were screened in 374 isolates of marine bacteria. Only two enzymes were found in all isolates while the majority of enzyme activities were present in a smaller proportion of the isolates. A cluster analysis demonstrated no significant correlation between taxonomy and enzyme function. However, there was evidence of co-occurrence of some enzyme activity in the same isolate. In this study marine Proteobacteria had a higher complement of enzymes with biodiscovery potential than Actinobacteria; this contrasts with the terrestrial environment where the Actinobacteria phylum is a proven source of enzymes with important industrial applications. In addition, a number of novel enzyme functions were more abundant in this marine culture collection than would be expected on the basis of knowledge from terrestrial bacteria. There is a strong case for future investigation of marine bacteria as a source for biodiscovery.

Isolation and initial characterization of a novel type of Baeyer-Villiger monooxygenase activity from a marine microorganism.

A novel type of Baeyer-Villiger monooxygenase (BVMO) has been found in a marine strain of Stenotrophomonas maltophila strain PML168 that was isolated from a temperate intertidal zone. The enzyme is able to use NADH as the source of reducing power necessary to accept the atom of diatomic oxygen not incorporated into the oxyfunctionalized substrate. Growth studies have establish that the enzyme is inducible, appears to serve a catabolic role, and is specifically induced by one or more unidentified components of seawater as well as various anthropogenic xenobiotic compounds. A blast search of the primary sequence of the enzyme, recovered from the genomic sequence of the isolate, has placed this atypical BVMO in the context of the several hundred known members of the flavoprotein monooxygenase superfamily. A particular feature of this BVMO lies in its truncated C-terminal domain, which results in a relatively small protein (357 amino acids; 38.4 kDa). In addition, metagenomic screening has been conducted on DNA recovered from an extensive range of marine environmental samples to gauge the relative abundance and distribution of similar enzymes within the global marine microbial community. Although low, abundance was detected in samples from many marine provinces, confirming the potential for biodiscovery in marine microorganisms.

Asymmetric Sulfoxidation Catalyzed by a Vanadium-Containing Bromoperoxidase.

A vanadium-containing bromoperoxidase (VBrPO) from the alga Corallina officinalis has been shown to catalyze the stereoselective oxidation of some aromatic bicyclic sulfides to the corresponding (S)-sulfoxides in high (up to 91%) ee. Hydrogen peroxide was found to have a large effect on the catalyzed reaction, most likely due to an inhibition of VBrPO. High optical and chemical yields were found to be favored by a continuous slow addition of hydrogen peroxide to keep a low excess. The reaction gives no overoxidation to sulfone, and its stereochemistry is the opposite as compared to that previously found with the heme-containing chloroperoxidase (CPO) from Caldariomyces fumago.