Cytochrome P450 enzymes from the metabolically diverse bacterium Rhodopseudomonas palustris

Bell, Stephen; Hoskins, Nicola; Feng, Xu; Caprotti, Domenico; Rao, Zihe; Wong, Luet‐Lok

doi:10.1016/j.bbrc.2006.01.133

Cited by 72 publications

(79 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This signal disappeared upon reduction, but no additional signal of a FeS cluster was found. In contrast, oxidation of Fdx2 with increasing amounts of K 3 [Fe(CN) 6 ] led to a considerable increase in the intensity of the [3Fe-4S] cluster signal, which displayed g factors of g 1 ϭ 2.020, g 2 ϭ 1.998, and g 3 ϭ 1.963. Under oxidizing conditions Fdx8 gave a signal at very low temperature (10 K) with a narrow line at g ϭ 2.021 and rather broad features at a higher field (Fig.…”

Section: ) Which Confirms Them To Be Non-[2fe-2s] Ferredoxins Althomentioning

confidence: 99%

“…Thus, it is desirable to identify the natural redox partners, especially if genomic sequence information is available. However, even then the identification of the correct interaction partners remains challenging because the encoding genes are frequently located at genomic loci distant to the cytochrome P450 genes (6,7). Interestingly, members of both the [2Fe-2S] and the non-[2Fe-2S] ferredoxins have been reported to sustain cytochrome P450 catalyzed reactions.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Genome Mining in Sorangium cellulosum So ce56

Ewen

Hannemann

Khatri

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

Myxobacteria, especially members of the genus Sorangium, are known for their biotechnological potential as producers of pharmaceutically valuable secondary metabolites. The biosynthesis of several of those myxobacterial compounds includes cytochrome P450 activity. Although class I cytochrome P450 enzymes occur wide-spread in bacteria and rely on ferredoxins and ferredoxin reductases as essential electron mediators, the study of these proteins is often neglected. Therefore, we decided to search in the Sorangium cellulosum So ce56 genome for putative interaction partners of cytochromes P450. In this work we report the investigation of eight myxobacterial ferredoxins and two ferredoxin reductases with respect to their activity in cytochrome P450 systems. Intriguingly, we found not only one, but two ferredoxins whose ability to sustain an endogenous So ce56 cytochrome P450 was demonstrated by CYP260A1-dependent conversion of nootkatone. Moreover, we could demonstrate that the two ferredoxins were able to receive electrons from both ferredoxin reductases. These findings indicate that S. cellulosum can alternate between different electron transport pathways to sustain cytochrome P450 activity. The cytochrome P450 (CYP)2 enzymes constitute a superfamily of external monooxygenases. The catalytic versatility of the family members explains their involvement in such diverse biological processes as biosynthesis of steroid hormones, carbon source assimilation, and metabolism of xenobiotics. In addition, cytochrome P450 enzymes have been reported to be involved in the biosynthesis of many pharmaceutically interesting secondary metabolites from a variety of microorganisms (1-4). Cytochromes P450 are usually dependent on an external electron donor. With respect to their electron transport system they can be divided into several classes, with class I (the mitochondrial/bacterial cytochrome P450 systems) being the predominant form in prokaryotes (5). In this system the electrons required for the enzymatic reaction originate from NAD(P)H and are delivered to the cytochrome P450 via a ferredoxin reductase and a ferredoxin. In a number of examples, the heterologous reconstitution of the electron transfer chain has been shown to be ineffective, if possible at all (5). Thus, it is desirable to identify the natural redox partners, especially if genomic sequence information is available. However, even then the identification of the correct interaction partners remains challenging because the encoding genes are frequently located at genomic loci distant to the cytochrome P450 genes (6, 7 To fulfill the role as electron mediator, the ferredoxin component of any given cytochrome P450 system has to be reduced. This reduction is achieved by a ferredoxin reductase, which in turn takes up electrons from NAD(P)H. The ferredoxin reductase is often the least characterized constituent of the cytochrome P450 system because these flavoproteins may be unstable (i.e. easily lose their cofactor) and usually show a relatively low level of expression (10...

show abstract

Section: ) Which Confirms Them To Be Non-[2fe-2s] Ferredoxins Althomentioning

confidence: 99%

mentioning

confidence: 99%

Genome Mining in Sorangium cellulosum So ce56

Ewen

Hannemann

Khatri

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Altering the substrate to a benzyl alcohol or a benzaldehyde both had a debilitative effect on substrate binding. [14,15] 4-Methoxybenzaldehyde induced a large spin state shift (80%) but the binding was three orders of magnitude weaker compared to 4-methoxybenzoic acid. The corresponding alcohol, 4-methoxybenzylalcohol, did not induce a significant spin state shift.…”

Section: Accepted M Manuscriptmentioning

confidence: 98%

“…We have also shown that 4-methoxybenzyl alcohol induced a minimal spin state shift on binding to CYP199A4. [14] The CYP199A4 catalysed turnover of 4-methoxybenzyl alcohol resulted in no product formation arising from substrate oxidation while 4-methoxybenzaldehyde generated 4-hydroxybenzaldehyde as the sole product but at a low turnover rate and coupling efficiency (Table 1). [15] The addition of 4-methoxybenzamide to CYP199A4 induced a 95% spin state shift.…”

Section: Substrate Bindingmentioning

confidence: 99%

“…[14,15] However, replacement of the methoxy moiety with alternative groups or the addition of small substituents at the 2-or 3-positions is tolerated. [14][15][16] For instance, CYP199A4 can bind and O-demethylate 2,4-and 3,4-dimethoxybenzoic acids with total regioselectivity at the 4-methoxy group to yield 2-methoxy-and 3-methoxy-4-hydroxybenzoic acids, respectively. [1,16] CYP199A4 has also…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

The importance of the benzoic acid carboxylate moiety for substrate recognition by CYP199A4 from Rhodopseudomonas palustris HaA2

Coleman

Chao

Voss

et al. 2016

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

Self Cite

View full text Add to dashboard Cite

The importance of the benzoic acid carboxylate moiety for substrate recognition by CYP199A4 from Rhodopseudomonas palustris HaA2, BBA -Proteins and Proteomics (2016), doi: 10.1016/j.bbapap.2016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Abstract BackgroundThe cytochrome P450 enzyme CYP199A4 can efficiently demethylate 4-methoxybenzoic acid. The substrate is positioned in the enzyme active site with the methoxy group ideally positioned for demethylation. This occurs through interactions of hydrophobic benzene ring with aromatic phenylalanine residues and the charged carboxylate group with polar and basic amino acids. MethodsIn vitro substrate binding and kinetic turnovers assays coupled with HPLC and GC-MS analysis and whole-cell oxidation turnovers. ResultsModification of the carboxylate group to an amide or aldehyde resulted in substrate binding, as judged by the almost total shift of the spin state to the high-spin form, but binding was three orders of magnitude weaker. Changing the carboxylate to phenol alcohol, ketone, ester and nitro groups and boronic, sulfinic and sulfonic acids resulted in a dramatic reduction in the binding affinity. Even phenylacetic acids were mediocre substrates for CYP199A4, despite maintaining a carboxylate group. The weaker binding of all of these substrates results in lower levels of turnover activity and product formation compared to 4-methoxybenzoic acid. ConclusionSubstrate binding to CYP199A4 is tightly regulated by interactions between the 4-methoxybenzoic acid and the amino acids in the active site. The benzoic acid carboxylate moiety is critical for optimal substrate binding and turnover activity with CYP199A4. General SignificanceAn understanding of how the CYP199A4 enzyme has evolved to be highly selective for para-substituted benzoic acids. This provides valuable insight into how other, as yet structurally uncharacterised, monooxygenase enzymes may bind benzoic acid substrates. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT3 Highlights CYP199A4 has high selectivity for the carboxylate group of 4-methoxybenzoic acid.This selectivity arises from interactions with arginine and serine residues.Alternative functional groups can bind to CYP199A4 but with low affinity.The activities of the in vitro enzyme assays can be extrapolated to whole-cell oxidation systems.The high regioselectivity for oxidation at the para-substituent is maintained.

show abstract