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.

Cloning of two gene clusters involved in the catabolism of 2,4-dinitrophenol by Paraburkholderia sp. strain KU-46 and characterization of the initial DnpAB enzymes and a two-component monooxygenases DnpC1C2.

Little is currently known about the metabolism of the industrial pollutant 2,4-dinitrophenol (DNP), particularly among gram-negative bacteria. In this study, we identified two non-contiguous genetic loci spanning 22 kb of Paraburkholderia (formerly Burkholderia) sp. strain KU-46. Additionally, we characterized four key initial genes (dnpA, dnpB, and dnpC1C2) responsible for DNP degradation, providing molecular and biochemical evidence for the degradation of DNP via the formation of 4-nitrophenol (NP), a pathway that is unique among DNP utilizing bacteria. Reverse transcription polymerase chain reaction (PCR) analysis indicated that dnpA, which encodes the initial hydride transferase, and dnpB which encodes a nitrite-eliminating enzyme, were induced by DNP and organized in an operon. Moreover, we purified DnpA and DnpB from recombinant Escherichia coli to demonstrate their effect on the transformation of DNP to NP through the formation of a hydride-Meisenheimer complex of DNP, designated as H--DNP. The function of DnpB appears new since all homologs of the DnpB sequences in the protein database are annotated as putative nitrate ABC transporter substrate-binding proteins. The gene cluster responsible for the degradation of DNP after NP formation was designated dnpC1C2DXFER, and DnpC1 and DnpC2 were functionally characterized as the FAD reductase and oxygenase components of the two-component DNP monooxygenase, respectively. By elucidating the hqdA1A2BCD gene cluster, we are now able to delineate the final degradation pathway of hydroquinone to ß-ketoadipate before it enters the tricarboxylic acid cycle.

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.

A two-component monooxygenase for continuous denitration and dechlorination of chlorinated 4-nitrophenol in Ensifer sp. strain 22-1.

The environmental fates of chlorinated 4-nitrophenols, 2,6-dichloro-4-nitrophenol (2,6-DCNP) and 2-chloro-4-nitrophenol (2C4NP), mediated via microbial catabolism have attracted great attention due to their high toxicity and persistence in the environment. In this study, a strain of Ensifer sp. 22-1 that was capable of degrading both 2,6-DCNP and 2C4NP was isolated from a halogenated aromatic-contaminated soil sample. A gene cluster cnpBADCERM was predicted to be involved in the catabolism of 2,6-DCNP and 2C4NP based on genome sequence analysis. A two-component monooxygenase CnpAB, composed of an oxygenase component (CnpA) and a reductase component (CnpB), was confirmed to catalyze the continuous denitration and dechlorination of 2,6-DCNP and 2C4NP to 6-chlorohydroxyquinol (6-CHQ) and hydroxyquinol (HQ), respectively. Knockout of cnpA resulted in the complete loss of the capacity for strain 22-1 to degrade 2,6-DCNP and 2C4NP. Homologous modeling and docking showed that Val155~Ala159, Phe206~Pro209 and Phe446~Arg461 of CnpA participated in the formation of the FAD-binding pocket, and Arg101, Val155 and Asn447 formed hydrogen bonds with 2,6-DCNP/2C4NP in the substrate-binding pocket. This work characterized a new two-component monooxygenase for 2,6-DCNP and 2C4NP, and enriched our understanding of the degradation mechanism of chlorinated nitrophenols (CNPs) by microorganisms.

Styrene monooxygenases, indole monooxygenases and related flavoproteins applied in bioremediation and biocatalysis.

Styrene and indole are naturally occurring compounds, which are also produced and processed by various chemical industries. Thus, it is not surprisingly that microorganisms evolved pathways to detoxify or even to utilize those compounds as carbon sources. Especially, among bacteria several routes are described specifically for the activation and degradation of styrene and indole. Respectively, the initial attack toward these compounds occurs via a flavin-dependent monooxygenase: styrene monooxygenase (SMO) or indole monooxygenase (IMO). In the first place, SMOs have been described to initiate a styrene specific degradation. These are in general two-component systems, whereas a small FAD-reductase (SMOB) delivers reduced FAD on the expense of NADH toward the monooxygenase (SMOA). Various modes of interaction are possible and for both mostly dimeric protein subunits structural data were reported. Thus, this flavoprotein monooxygenase-especially the one from Pseudomonas putida S12 can be seen as the prototype of this class of enzymes. In the course of describing related members of this enzyme family some remarkable findings were made. For example, self-sufficient fusion proteins have been reported as well as enzymes, which could not be assigned to a styrene metabolic activity, rather to indole conversion. Later it was found that this flavoprotein group can be separated at least into two subgroups: styrene and indole monooxygenases. And both enzymes rely on a FAD-reductase to obtain the reduced cofactor (FADred), which is employed to activate molecular oxygen toward hydroperoxy-FAD, which allows substrate epoxidation and the formation of hydroxy-FAD, which finally yields H2O and oxidized FAD.

Bacterial luciferase: Molecular mechanisms and applications.

Bacterial luciferase is a flavin-dependent monooxygenase which is remarkable for its distinctive feature in transforming chemical energy to photons of visible light. The bacterial luciferase catalyzes bioluminescent reaction using reduced flavin mononucleotide, long-chain aldehyde and oxygen to yield oxidized flavin, corresponding acid, water and light at λmax around 490nm. The enzyme comprises of two non-identical α and ß subunits, where α subunit is a catalytic center and ß subunit is crucially required for maintaining catalytic function of the α subunit. The crystal structure with FMN bound and mutagenesis studies have assigned a number of amino acid residues that are important in coordinating critical reactions and stabilizing intermediates to attain optimum reaction efficiency. The enzyme achieves monooxygenation by generating C4a-hydroperoxyflavin intermediate that later changes its protonation status to become C4a-peroxyflavin, which is necessary for the nucleophilic attacking with aldehyde substrate. The decomposing of C4a-peroxyhemiacetal produces excited C4a-hydroxyflavin and acid product. The chemical basis regrading bioluminophore generation in Lux reaction remains an inconclusive issue. However, current data can, at least, demonstrate the involvement of electron transfer to create radical molecules which is the key step in this mechanism. Lux is a self-sufficient bioluminescent system in which all substrates can be recycled and produced by a group of enzymes from the lux operon. This makes Lux distinctively advantageous over other luciferases for reporter enzyme application. The progression of understanding of Lux catalysis is beneficial to improve light emitting efficiency in order to expand the robustness of Lux application.

Microbial degradation kinetics and molecular mechanism of 2,6-dichloro-4-nitrophenol by a Cupriavidus strain.

2,6-Dichloro-4-nitrophenol (2,6-DCNP) is an emerging chlorinated nitroaromatic pollutant, and its fate in the environment is an important question. However, microorganisms with the ability to utilize 2,6-DCNP have not been reported. In this study, Cupriavidus sp. CNP-8 having been previously reported to degrade various halogenated nitrophenols, was verified to be also capable of degrading 2,6-DCNP. Biodegradation kinetics assay showed that it degraded 2,6-DCNP with the specific growth rate of 0.124 h-1, half saturation constant of 0.038 mM and inhibition constant of 0.42 mM. Real-time quantitative PCR analyses indicated that the hnp gene cluster was involved in the catabolism of 2,6-DCNP. The hnpA and hnpB gene products were purified to homogeneity by Ni-NTA chromatography. Enzymatic assays showed that HnpAB, a FAD-dependent two-component monooxygenase, converted 2,6-DCNP to 6-chlorohydroxyquinol with a Km of 3.9 ± 1.4 µM and a kcat/Km of 0.12 ± 0.04 µΜ-1 min-1. As the oxygenase component encoding gene, hnpA is necessary for CNP-8 to grow on 2,6-DCNP by gene knockout and complementation. The phylogenetic analysis showed that the hnp cluster originated from the cluster involved in the catabolism of chlorophenols rather than nitrophenols. To our knowledge, CNP-8 is the first bacterium with the ability to utilize 2,6-DCNP, and this study fills a gap in the microbial degradation mechanism of this pollutant at the molecular, biochemical and genetic levels. Moreover, strain CNP-8 could degrade three chlorinated nitrophenols rapidly from the synthetic wastewater, indicating its potential in the bioremediation of chlorinated nitrophenols polluted environments.

Molecular and biochemical characterization of 2-chloro-4-nitrophenol degradation via the 1,2,4-benzenetriol pathway in a Gram-negative bacterium.

2-Chloro-4-nitrophenol (2C4NP) is the most common chlorinated nitrophenol pollutant, and its environmental fate is of great concern. Cupriavidus sp. CNP-8, a Gram-negative bacterium, has been reported to degrade 2C4NP via the 1,2,4-benzenetriol (BT) pathway, significantly different from the (chloro)hydroquinone pathways reported in all other Gram-negative 2C4NP-utilizers. Herein, the BT pathway of the catabolism of 2C4NP in this strain was characterized at the molecular, biochemical, and genetic levels. The hnp gene cluster was suspected to be involved in the catabolism of 2C4NP because the hnp genes are significantly upregulated in the 2C4NP-induced strain CNP-8 compared to the uninduced strain. HnpAB, a two-component FAD-dependent monooxygenase, catalyzes the conversion of 2C4NP to BT via chloro-1,4-benzoquinone, with a Km of 2.7 ± 1.1 µΜ and a kcat/Km of 0.17 ± 0.03 µΜ-1 min-1. hnpA is necessary for strain CNP-8 to utilize 2C4NP in vivo. HnpC, a BT 1,2-dioxygenase, was proved to catalyze BT ring-cleavage with formation of maleylacetate by HPLC-MS analysis. Phylogenetic analysis indicated that HnpA likely has different evolutionary origin compared to other functionally identified 2C4NP monooxygenases. To our knowledge, this is the first report revealing the catabolic mechanism of 2C4NP via the BT pathway in a Gram-negative bacterium, increasing our knowledge of the catabolic diversity for microbial 2C4NP degradation at the molecular and biochemical level.

Investigations of two-component flavin-dependent monooxygenase systems.

Bacterial two-component flavin-dependent monooxygenase systems catalyze the oxidation of diverse metabolic reactions. There are several shared mechanistic features in the two-component monooxygenase systems that differ from canonical monooxygenase enzymes. The flavin reductases catalyze the reductive half-reaction, and the reduced flavin is transferred to the monooxygenase enzyme. The oxidative half-reaction catalyzed by the monooxygenase enzyme has been proposed to occur through the formation of a (hydro)peroxyflavin intermediate. In some two-component flavin-dependent systems the mechanism of flavin transfer involves protein-protein interactions between the flavin reductase and monooxygenase enzyme. Methods are presented that provide an alternative approach from flavin-bound monooxygenases to evaluate the kinetic properties and flavin transfer mechanism of the two-component flavin-dependent monooxygenase systems.

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.

VpStyA1/VpStyA2B of Variovorax paradoxus EPS: An Aryl Alkyl Sulfoxidase Rather than a Styrene Epoxidizing Monooxygenase.

Herein we describe the first representative of an E2-type two-component styrene monooxygenase of proteobacteria. It comprises a single epoxidase protein (VpStyA1) and a two domain protein (VpStyA2B) harboring an epoxidase (A2) and a FAD-reductase (B) domain. It was annotated as VpStyA1/VpStyA2B of Variovorax paradoxus EPS. VpStyA2B serves mainly as NADH:FAD-oxidoreductase. A Km of 33.6 ± 4.0 µM for FAD and a kcat of 22.3 ± 1.1 s-1 were determined and resulted in a catalytic efficiency (kcatKm-1) of 0.64 s-1 µM-1. To investigate its NADH:FAD-oxidoreductase function the linker between A2- and B-domain (AREAV) was mutated. One mutant (AAAAA) showed 18.7-fold higher affinity for FAD (kcatKm-1 of 5.21 s-1 µM-1) while keeping wildtype NADH-affinity and -oxidation activity. Both components, VpStyA2B and VpStyA1, showed monooxygenase activity on styrene of 0.14 U mg-1 and 0.46 U mg-1, as well as on benzyl methyl sulfide of 1.62 U mg-1 and 3.11 U mg-1, respectively. The high sulfoxidase activity was the reason to test several thioanisole-like substrates in biotransformations. VpStyA1 showed high substrate conversions (up to 95% in 2 h) and produced dominantly (S)-enantiomeric sulfoxides of all tested substrates. The AAAAA-mutant showed a 1.6-fold increased monooxygenase activity. In comparison, the GQWCSQY-mutant did neither show monooxygenase nor efficient FAD-reductase activity. Hence, the linker between the two domains of VpStyA2B has effects on the reductase as well as on the monooxygenase performance. Overall, this monooxygenase represents a promising candidate for biocatalyst development and studying natural fusion proteins.

Hydroxylation of 4-hydroxyphenylethylamine derivatives by R263 variants of the oxygenase component of p-hydroxyphenylacetate-3-hydroxylase.

p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to yield 3,4-dihydroxyphenylacetate (DHPA). In this study, we investigated whether variants of the oxygenase component (C2) could catalyze hydroxylation of 4-hydroxyphenylethylamines to synthesize catecholamine derivatives. Single turnover product analysis showed that the R263D variant can catalyze hydroxylation of tyramine to form dopamine with the highest yield (57%). The enzyme was also found to have dual substrate charge specificity because it can also maintain reasonable hydroxylation efficiency of HPA (86%). This property is different from the R263E variant, which can hydroxylate HPA (73%) but not tyramine. The R263A variant can hydroxylate HPA (72%) and tyramine to a small extent (7%). Stopped-flow experiments indicated that tyramine and HPA prefer binding to R263D after C4a-hydroperoxy-FMN formation, while tyramine cannot bind to the wild-type or R263E enzymes. Data also indicate that the hydroxylation rate constant is the rate-limiting step. The R263D variant was used as a starting enzyme for further mutation to obtain other variants for the synthesis of additional catecholamine drugs. The R263D/Y398D double mutant enzyme showed interesting results in that it was able to catalyze the hydroxylation of octopamine to form norepinephrine. However, the enzyme still lacked stereo-selectivity in its reaction.