Tailoring an alien ferredoxin to support native-like P450 monooxygenase activity

Bell, Stephen; McMillan, James H. C.; Yorke, Jake A.; Kavanagh, Eoin; Johnson, Eachan O. D.; Wong, Luet‐Lok

doi:10.1039/c2cc35968e

Cited by 31 publications

(41 citation statements)

References 19 publications

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“…A lot of examples have demonstrated that the interactions between P450s and Fd proteins are significant for P450 activity. For instance, Bell et al tailored an non-physiological Fd to support native-like P450 activity through engineering the surface residues involved in the interaction between Fd and P450 [40]; P450 105D5 from Streptomyces coelicolor A3(2) is very selective among S. coelicolor Fd proteins, but Fd could interact with the surrogate FNRs from P. putida and spinach [22]; With S. griseolus P450 105A1 and 105B1, either of two Fd proteins could reconstitute P450 activity, but each Fd worked at least somewhat faster with its cognate P450 [41]. …”

Section: Resultsmentioning

confidence: 99%

Conversion of fatty aldehydes into alk (a/e)nes by in vitroreconstituted cyanobacterial aldehyde-deformylating oxygenase with the cognate electron transfer system

Zhang

Lü

2013

Biotechnol Biofuels

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BackgroundBiosynthesis of fatty alk(a/e)ne in cyanobacteria has been considered as a potential basis for the sunlight-driven and carbon-neutral bioprocess producing advanced solar biofuels. Aldehyde-deformylating oxygenase (ADO) is a key enzyme involved in that pathway. The heterologous or chemical reducing systems were generally used in in vitro ADO activity assay. The cognate electron transfer system from cyanobacteria to support ADO activity is still unknown.ResultsWe identified the potential endogenous reducing system including ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) to support ADO activity in Synechococcus elongatus PCC7942. ADO (Synpcc7942_1593), FNR (SynPcc7942_0978), and Fd (SynPcc7942_1499) from PCC7942 were cloned, overexpressed, purified, and characterized. ADO activity was successfully supported with the endogenous electron transfer system, which worked more effectively than the heterologous and chemical ones. The results of the hybrid Fd/FNR reducing systems demonstrated that ADO was selective against Fd. And it was observed that the cognate reducing system produced less H2O2 than the heterologous one by 33% during ADO-catalyzed reactions. Importantly, kcat value of ADO 1593 using the homologous Fd/FNR electron transfer system is 3.7-fold higher than the chemical one.ConclusionsThe cognate electron transfer system from cyanobacteria to support ADO activity was identified and characterized. For the first time, ADO was functionally in vitro reconstituted with the endogenous reducing system from cyanobacteria, which supported greater activity than the surrogate and chemical ones, and produced less H2O2 than the heterologous one. The identified Fd/FNR electron transfer system will be potentially useful for improving ADO activity and further enhancing the biosynthetic efficiency of hydrocarbon biofuels in cyanobacteria.

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Section: Resultsmentioning

confidence: 99%

Conversion of fatty aldehydes into alk (a/e)nes by in vitroreconstituted cyanobacterial aldehyde-deformylating oxygenase with the cognate electron transfer system

Zhang

Lü

2013

Biotechnol Biofuels

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“…However, a detailed analysis of the substrate binding and enzyme kinetics was not presented as the experiments were performed by using a whole‐cell oxidation system . Knowledge of the critical enzyme–substrate interactions is of paramount importance to facilitate efforts to engineer enzyme systems rationally to achieve the turnover numbers required for large‐scale synthetic applications ,,. Previously, we have shown that the R243T, R92E and S95V mutants all have a significant debilitative effect on 4‐methoxybenzoic acid binding to CYP199A4 and reduce the activity of the enzyme …”

Section: Introductionmentioning

confidence: 99%

Modification of an Enzyme Biocatalyst for the Efficient and Selective Oxidative Demethylation of para‐Substituted Benzene Derivatives

et al. 2016

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The bacterial CYP199A4 enzyme is able to oxidise a narrow range of aromatic acids, which includes 4‐methoxybenzoic acid, efficiently. A serine 244 to aspartate variant was identified with enhanced activity for a wide range of para‐methoxy‐substituted benzenes. Substrates in which the acidic benzoic acid moiety is replaced with a phenol and the amide, aldehyde and bromide analogues were all oxidised with high activity by the S244D mutant (product formation rate >600 nmol nmolCYP−1 min−1) with turnover numbers of up to 20 000. If the carboxylate moiety was modified to a nitro, ketone, boronic acid, hydroxymethyl or nitrile group, these substrates were also oxidised at a significantly higher activity by S244D than the wild‐type enzyme. 3,4‐Dimethoxybenzaldehyde was demethylated selectively and oxidatively to 3‐methoxy‐4‐hydroxybenzaldehyde by the S244D mutant 84‐fold more rapidly than with the wild‐type enzyme. CYP199A4 would have applications in the catalytic regioselective oxidative demethylation of suitably substituted benzene substrates under mild conditions and in the presence of more oxidatively sensitive functional groups, such as an aldehyde.

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“…Spectroelectrochemical measurements at pH 7.4 indicate that the Fe(III)Fe(III)/Fe(III)Fe(II) reduction of Pux occurs at −0.25 V, whereas that of PuxB occurs at −0.29 V (V, vs. NHE) [60].…”

Section: Rhodopseudomonas Palustrismentioning

confidence: 99%

The competition between chemistry and biology in assembling iron–sulfur derivatives. Molecular structures and electrochemistry. Part II. {[Fe2S2](SγCys)4} proteins

Zanello¹

2014

Coordination Chemistry Reviews

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Tailoring an alien ferredoxin to support native-like P450 monooxygenase activity

Cited by 31 publications

References 19 publications

Conversion of fatty aldehydes into alk (a/e)nes by in vitroreconstituted cyanobacterial aldehyde-deformylating oxygenase with the cognate electron transfer system

Conversion of fatty aldehydes into alk (a/e)nes by in vitroreconstituted cyanobacterial aldehyde-deformylating oxygenase with the cognate electron transfer system

Modification of an Enzyme Biocatalyst for the Efficient and Selective Oxidative Demethylation of para‐Substituted Benzene Derivatives

The competition between chemistry and biology in assembling iron–sulfur derivatives. Molecular structures and electrochemistry. Part II. {[Fe2S2](SγCys)4} proteins

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