Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase

Wu, Sheng-Yi; Rothery, Richard A.; Weiner, Joël H.

doi:10.1074/jbc.m115.665422

Cited by 23 publications

(23 citation statements)

References 62 publications

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“…So far, a number of DMSOR family members have been produced by homologously or heterologously expressing their corresponding genes ( 27 – 34 ). In contrast, attempts to produce the phylogenetically related, complex heterotrimeric molybdenum-iron-sulfur/heme b-containing alkyl chain hydroxylases of the class II DMSOR family in an active form have failed so far.…”

Section: Discussionmentioning

confidence: 99%

Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D ₃ Synthesis

Jacoby

Eipper

Warnke

et al. 2018

mBio

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Side chain-containing steroids are ubiquitous constituents of biological membranes that are persistent to biodegradation. Aerobic, steroid-degrading bacteria employ oxygenases for isoprenoid side chain and tetracyclic steran ring cleavage. In contrast, a Mo-containing steroid C-25 dehydrogenase (S25DH) of the dimethyl sulfoxide (DMSO) reductase family catalyzes the oxygen-independent hydroxylation of tertiary C-25 in the anaerobic, cholesterol-degrading bacterium Sterolibacterium denitrificans. Its genome contains eight paralogous genes encoding active site α-subunits of putative S25DH-like proteins. The difficult enrichment of labile, oxygen-sensitive S25DH from the wild-type bacteria and the inability of its active heterologous production have largely hampered the study of S25DH-like gene products. Here we established a heterologous expression platform for the three structural genes of S25DH subunits together with an essential chaperone in the denitrifying betaproteobacterium Thauera aromatica K172. Using this system, S25DH1 and three isoenzymes (S25DH2, S25DH3, and S25DH4) were overproduced in a soluble, active form allowing a straightforward purification of nontagged αβγ complexes. All S25DHs contained molybdenum, four [4Fe-4S] clusters, one [3Fe-4S] cluster, and heme B and catalyzed the specific, water-dependent C-25 hydroxylations of various 4-en-3-one forms of phytosterols and zoosterols. Crude extracts from T. aromatica expressing genes encoding S25DH1 catalyzed the hydroxylation of vitamin D3 (VD3) to the clinically relevant 25-OH-VD3 with >95% yield at a rate 6.5-fold higher than that of wild-type bacterial extracts; the specific activity of recombinant S25DH1 was twofold higher than that of wild-type enzyme. These results demonstrate the potential application of the established expression platform for 25-OH-VD3 synthesis and pave the way for the characterization of previously genetically inaccessible S25DH-like Mo enzymes of the DMSO reductase family.

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Section: Discussionmentioning

confidence: 99%

Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D ₃ Synthesis

Jacoby

Eipper

Warnke

et al. 2018

mBio

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“…Intriguingly, both pterins and flavins undergo two-electron oxidoreduction reactions ( Figure 7 ). Noteworthily, even in extant molybdoenzymes, redox properties are strongly influenced by the H-bonding network surrounding the pterin moiety ( Wu et al, 2015 ; Duval et al, 2016 ) just as is the case for isoalloxazine molecule in flavoenzymes. Thus, while molydopterin itself was retained for redox ranges inaccessible to the flavins, the flavoproteins themselves may have evolved to stand in for the absent molybdenum anion in many of its other former roles, especially perhaps in the redox bifurcation of electrons.…”

Section: On the Evolutionary Emergence Of Electron Bifurcationmentioning

confidence: 99%

On the Natural History of Flavin-Based Electron Bifurcation

et al. 2018

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Electron bifurcation is here described as a special case of the continuum of electron transfer reactions accessible to two-electron redox compounds with redox cooperativity. We argue that electron bifurcation is foremost an electrochemical phenomenon based on (a) strongly inverted redox potentials of the individual redox transitions, (b) a high endergonicity of the first redox transition, and (c) an escapement-type mechanism rendering completion of the first electron transfer contingent on occurrence of the second one. This mechanism is proposed to govern both the traditional quinone-based and the newly discovered flavin-based versions of electron bifurcation. Conserved and variable aspects of the spatial arrangement of electron transfer partners in flavoenzymes are assayed by comparing the presently available 3D structures. A wide sample of flavoenzymes is analyzed with respect to conserved structural modules and three major structural groups are identified which serve as basic frames for the evolutionary construction of a plethora of flavin-containing redox enzymes. We argue that flavin-based and other types of electron bifurcation are of primordial importance to free energy conversion, the quintessential foundation of life, and discuss a plausible evolutionary ancestry of the mechanism.

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“…In principle, one Mo-ion is coordinated with two pyranopterins and interacts with cys-(NAP) or ser-or asp-residues (NAR) (pI 5 5.1, 5.7 and 2.8 respectively) (Moura et al, 2004). Two conserved his-residues in the proximity of the pyranopterin rings appear to be of significance for the catalytic activity of NAR (Bertero et al, 2003;Wu et al, 2015). In their protonated form the hisresidues stabilize the pyranopterin rings and the Mo(V) intermediate during catalysis.…”

Section: The Effect Of Ph On Individual Enzymes Involved In N-conversionmentioning

confidence: 99%

“…In their protonated form the hisresidues stabilize the pyranopterin rings and the Mo(V) intermediate during catalysis. On loss of an overall positive charge, the midterm potential of the molybdenum ion decreases, which is directly correlated with enzyme activity pH dependency of N-converting enzymatic processes, pathways and microbes 1629 (Wu et al, 2015). Therefore, an increase of pH appears to directly transfer into a loss of catalytic turnover of NAR.…”

Section: The Effect Of Ph On Individual Enzymes Involved In N-conversionmentioning

confidence: 99%

The pH dependency of N‐converting enzymatic processes, pathways and microbes: effect on net N₂O production

Blum

et al. 2018

Environmental Microbiology

101

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Nitrous oxide (N O) is emitted during microbiological nitrogen (N) conversion processes, when N O production exceeds N O consumption. The magnitude of N O production vs. consumption varies with pH and controlling net N O production might be feasible by choice of system pH. This article reviews how pH affects enzymes, pathways and microorganisms that are involved in N-conversions in water engineering applications. At a molecular level, pH affects activity of cofactors and structural elements of relevant enzymes by protonation or deprotonation of amino acid residues or solvent ligands, thus causing steric changes in catalytic sites or proton/electron transfer routes that alter the enzymes' overall activity. Augmenting molecular information with, e.g., nitritation or denitrification rates yields explanations of changes in net N O production with pH. Ammonia oxidizing bacteria are of highest relevance for N O production, while heterotrophic denitrifiers are relevant for N O consumption at pH > 7.5. Net N O production in N-cycling water engineering systems is predicted to display a 'bell-shaped' curve in the range of pH 6.0-9.0 with a maximum at pH 7.0-7.5. Net N O production at acidic pH is dominated by N O production, whereas N O consumption can outweigh production at alkaline pH. Thus, pH 8.0 may be a favourable pH set-point for water treatment applications regarding net N O production.

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Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase

Cited by 23 publications

References 62 publications

Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D ₃ Synthesis

Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D ₃ Synthesis

On the Natural History of Flavin-Based Electron Bifurcation

The pH dependency of N‐converting enzymatic processes, pathways and microbes: effect on net N₂O production

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Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase

Cited by 23 publications

References 62 publications

Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D 3 Synthesis

Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D 3 Synthesis

On the Natural History of Flavin-Based Electron Bifurcation

The pH dependency of N‐converting enzymatic processes, pathways and microbes: effect on net N2O production

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Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D ₃ Synthesis

Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D ₃ Synthesis

The pH dependency of N‐converting enzymatic processes, pathways and microbes: effect on net N₂O production