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Mycobacterial F420H2-Dependent Reductases Promiscuously Reduce Diverse Compounds through a Common Mechanism.

ABSTRACT: An unusual aspect of actinobacterial metabolism is the use of the redox cofactor F420. Studies have shown that actinobacterial F420H2-dependent reductases promiscuously hydrogenate diverse organic compounds in biodegradative and biosynthetic processes. These enzymes therefore represent promising candidates for next-generation industrial biocatalysts. In this work, we undertook the first broad survey of these enzymes as potential industrial biocatalysts by exploring the extent, as well as mechanistic and structural bases, of their substrate promiscuity. We expressed and purified 11 enzymes from seven subgroups of the flavin/deazaflavin oxidoreductase (FDOR) superfamily (A1, A2, A3, B1, B2, B3, B4) from the model soil actinobacterium Mycobacterium smegmatis. These enzymes reduced compounds from six chemical classes, including fundamental monocycles such as a cyclohexenone, a dihydropyran, and pyrones, as well as more complex quinone, coumarin, and arylmethane compounds. Substrate range and reduction rates varied between the enzymes, with the A1, A3, and B1 groups exhibiting greatest promiscuity. Molecular docking studies suggested that structurally diverse compounds are accommodated in the large substrate-binding pocket of the most promiscuous FDOR through hydrophobic interactions with conserved aromatic residues and the isoalloxazine headgroup of F420H2. Liquid chromatography-mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS) analysis of derivatized reaction products showed reduction occurred through a common mechanism involving hydride transfer from F420H- to the electron-deficient alkene groups of substrates. Reduction occurs when the hydride donor (C5 of F420H-) is proximal to the acceptor (electrophilic alkene of the substrate). These findings suggest that engineered actinobacterial F420H2-dependent reductases are promising novel biocatalysts for the facile transformation of a wide range of ?,?-unsaturated compounds.

SUBMITTER: Greening C

PROVIDER: S-EPMC5449967 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Mycobacterial F420H2-Dependent Reductases Promiscuously Reduce Diverse Compounds through a Common Mechanism.

Greening Chris C Jirapanjawat Thanavit T Afroze Shahana S Ney Blair B Scott Colin C Pandey Gunjan G Lee Brendon M BM Russell Robyn J RJ Jackson Colin J CJ Oakeshott John G JG Taylor Matthew C MC Warden Andrew C AC

Frontiers in microbiology 20170531

An unusual aspect of actinobacterial metabolism is the use of the redox cofactor F420. Studies have shown that actinobacterial F420H2-dependent reductases promiscuously hydrogenate diverse organic compounds in biodegradative and biosynthetic processes. These enzymes therefore represent promising candidates for next-generation industrial biocatalysts. In this work, we undertook the first broad survey of these enzymes as potential industrial biocatalysts by explori ...[more]

PMID: 28620367

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Project description:Coenzyme F420 is a microbial redox cofactor that mediates diverse physiological functions and is increasingly used for biocatalytic applications. Recently, diversified biosynthetic routes to F420 and the discovery of a derivative, 3PG-F420, were reported. 3PG-F420 is formed via activation of 3-phospho-d-glycerate (3-PG) by CofC, but the structural basis of substrate binding, its evolution, as well as the role of CofD in substrate selection remained elusive. Here, we present a crystal structure of the 3-PG-activating CofC from Mycetohabitans sp. B3 and define amino acids governing substrate specificity. Site-directed mutagenesis enabled bidirectional switching of specificity and thereby revealed the short evolutionary trajectory to 3PG-F420 formation. Furthermore, CofC stabilized its product, thus confirming the structure of the unstable molecule and revealing its binding mode. The CofD enzyme was shown to significantly contribute to the selection of related intermediates to control the specificity of the combined biosynthetic CofC/D step. These results imply the need to change the design of combined CofC/D activity assays. Taken together, this work presents novel mechanistic and structural insights into 3PG-F420 biosynthesis and evolution and opens perspectives for the discovery and enhanced biotechnological production of coenzyme F420 derivatives in the future. IMPORTANCE The microbial cofactor F420 is crucial for processes like methanogenesis, antibiotics biosynthesis, drug resistance, and biocatalysis. Recently, a novel derivative of F420 (3PG-F420) was discovered, enabling the production and use of F420 in heterologous hosts. By analyzing the crystal structure of a CofC homolog whose substrate choice leads to formation of 3PG-F420, we defined amino acid residues governing the special substrate selectivity. A diagnostic residue enabled reprogramming of the substrate specificity, thus mimicking the evolution of the novel cofactor derivative. Furthermore, a labile reaction product of CofC was revealed that has not been directly detected so far. CofD was shown to provide another layer of specificity of the combined CofC/D reaction, thus controlling the initial substrate choice of CofC. The latter finding resolves a current debate in the literature about the starting point of F420 biosynthesis in various organisms.

Project description:Anaerobic methanotrophic archaea (ANME), which oxidize methane in marine sediments through syntrophic associations with sulfate-reducing bacteria, carry homologs of coenzyme F420-dependent sulfite reductase (Fsr) of Methanocaldococcus jannaschii, a hyperthermophilic methanogen from deep-sea hydrothermal vents. M. jannaschii Fsr (MjFsr) and ANME-Fsr belong to two phylogenetically distinct groups, FsrI and FsrII, respectively. MjFsrI reduces sulfite to sulfide with reduced F420 (F420H2), protecting methyl coenzyme M reductase (Mcr), an essential enzyme for methanogens, from sulfite inhibition. However, the function of FsrIIs in ANME, which also rely on Mcr and live in sulfidic environments, is unknown. We have determined the catalytic properties of FsrII from a member of ANME-2c. Since ANME remain to be isolated, we expressed ANME2c-FsrII in a closely related methanogen, Methanosarcina acetivorans. Purified recombinant FsrII contained siroheme, indicating that the methanogen, which lacks a native sulfite reductase, produced this coenzyme. Unexpectedly, FsrII could not reduce sulfite or thiosulfate with F420H2. Instead, it acted as an F420H2-dependent nitrite reductase (FNiR) with physiologically relevant Km values (nitrite, 5 μM; F420H2, 14 μM). From kinetic, thermodynamic, and structural analyses, we hypothesize that in FNiR, F420H2-derived electrons are delivered at the oxyanion reduction site at a redox potential that is suitable for reducing nitrite (E0' [standard potential], +440 mV) but not sulfite (E0', -116 mV). These findings and the known nitrite sensitivity of Mcr suggest that FNiR may protect nondenitrifying ANME from nitrite toxicity. Remarkably, by reorganizing the reductant processing system, Fsr transforms two analogous oxyanions in two distinct archaeal lineages with different physiologies and ecologies. IMPORTANCE Coenzyme F420-dependent sulfite reductase (Fsr) protects methanogenic archaea inhabiting deep-sea hydrothermal vents from the inactivation of methyl coenzyme M reductase (Mcr), one of their essential energy production enzymes. Anaerobic methanotrophic archaea (ANME) that oxidize methane and rely on Mcr, carry Fsr homologs that form a distinct clade. We show that a member of this clade from ANME-2c functions as F420-dependent nitrite reductase (FNiR) and lacks Fsr activity. This specialization arose from a distinct feature of the reductant processing system and not the substrate recognition element. We hypothesize FNiR may protect ANME Mcr from inactivation by nitrite. This is an example of functional specialization within a protein family that is induced by changes in electron transfer modules to fit an ecological need.

Dataset Information

Mycobacterial F420H2-Dependent Reductases Promiscuously Reduce Diverse Compounds through a Common Mechanism.

Publications

Mycobacterial F<sub>420</sub>H<sub>2</sub>-Dependent Reductases Promiscuously Reduce Diverse Compounds through a Common Mechanism.

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