The dimeric form of flavocytochrome P450 BM3 is catalytically functional as a fatty acid hydroxylase

Rajasekhar Neeli; Hazel M. Girvan; Andrew Lawrence; Martin J. Warren; David Leys; Nigel S. Scrutton; Andrew W. Munro

doi:10.1016/j.febslet.2005.09.023

The dimeric form of flavocytochrome P450 BM3 is catalytically functional as a fatty acid hydroxylase

Rajasekhar Neeli, Hazel M. Girvan, Andrew Lawrence, Martin J. Warren, David Leys, Nigel S. Scrutton, Andrew W. Munro

Research output: Contribution to journal › Article › peer-review

110 Citations (Scopus)

Abstract

In the model P450 BM3 system, the P450 is fused to its diflavin reductase partner in a single polypeptide. BM3 dimerizes in solution, but the catalytic relevance of the phenomenon was hitherto unknown. We show that BM3 fatty acid hydroxylase specific activity decreases sharply at low enzyme concentrations, consistent with separation of active dimer into inactive monomer. Reductase-dependent specific activities are maintained or enhanced at low concentration, suggesting inter-flavin electron transfer is unaffected. Fatty acid oxidation is reconstituted by mixing inactive oxygenase (A264H) and FMN-depleted (G570D) mutants, demonstrating that inter-monomer (FMN ₁-to-heme₂) electron transfer supports oxygenase activity in the BM3 dimer.

Original language	English
Pages (from-to)	5582-5588
Number of pages	7
Journal	FEBS Letters
Volume	579
Issue number	25
Early online date	28 Sep 2005
DOIs	https://doi.org/10.1016/j.febslet.2005.09.023
Publication status	Published - 24 Oct 2005
Externally published	Yes

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title = "The dimeric form of flavocytochrome P450 BM3 is catalytically functional as a fatty acid hydroxylase",

abstract = "In the model P450 BM3 system, the P450 is fused to its diflavin reductase partner in a single polypeptide. BM3 dimerizes in solution, but the catalytic relevance of the phenomenon was hitherto unknown. We show that BM3 fatty acid hydroxylase specific activity decreases sharply at low enzyme concentrations, consistent with separation of active dimer into inactive monomer. Reductase-dependent specific activities are maintained or enhanced at low concentration, suggesting inter-flavin electron transfer is unaffected. Fatty acid oxidation is reconstituted by mixing inactive oxygenase (A264H) and FMN-depleted (G570D) mutants, demonstrating that inter-monomer (FMN 1-to-heme2) electron transfer supports oxygenase activity in the BM3 dimer.",

keywords = "Cytochrome P450, Dimerization, Electron transfer, Enzymology, Fatty acids, Oxygenase, BM3, flavocytochrome P450 BM3 from Bacillus megaterium, CO, Carbon monoxide, AUC, analytical ultracentrifugation, eT, NOS, Nitric oxide synthase",

author = "Rajasekhar Neeli and Girvan, {Hazel M.} and Andrew Lawrence and Warren, {Martin J.} and David Leys and Scrutton, {Nigel S.} and Munro, {Andrew W.}",

year = "2005",

month = oct,

day = "24",

doi = "10.1016/j.febslet.2005.09.023",

language = "English",

volume = "579",

pages = "5582--5588",

journal = "FEBS Letters",

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publisher = "Elsevier",

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TY - JOUR

T1 - The dimeric form of flavocytochrome P450 BM3 is catalytically functional as a fatty acid hydroxylase

AU - Neeli, Rajasekhar

AU - Girvan, Hazel M.

AU - Lawrence, Andrew

AU - Warren, Martin J.

AU - Leys, David

AU - Scrutton, Nigel S.

AU - Munro, Andrew W.

PY - 2005/10/24

Y1 - 2005/10/24

N2 - In the model P450 BM3 system, the P450 is fused to its diflavin reductase partner in a single polypeptide. BM3 dimerizes in solution, but the catalytic relevance of the phenomenon was hitherto unknown. We show that BM3 fatty acid hydroxylase specific activity decreases sharply at low enzyme concentrations, consistent with separation of active dimer into inactive monomer. Reductase-dependent specific activities are maintained or enhanced at low concentration, suggesting inter-flavin electron transfer is unaffected. Fatty acid oxidation is reconstituted by mixing inactive oxygenase (A264H) and FMN-depleted (G570D) mutants, demonstrating that inter-monomer (FMN 1-to-heme2) electron transfer supports oxygenase activity in the BM3 dimer.

AB - In the model P450 BM3 system, the P450 is fused to its diflavin reductase partner in a single polypeptide. BM3 dimerizes in solution, but the catalytic relevance of the phenomenon was hitherto unknown. We show that BM3 fatty acid hydroxylase specific activity decreases sharply at low enzyme concentrations, consistent with separation of active dimer into inactive monomer. Reductase-dependent specific activities are maintained or enhanced at low concentration, suggesting inter-flavin electron transfer is unaffected. Fatty acid oxidation is reconstituted by mixing inactive oxygenase (A264H) and FMN-depleted (G570D) mutants, demonstrating that inter-monomer (FMN 1-to-heme2) electron transfer supports oxygenase activity in the BM3 dimer.

KW - Cytochrome P450

KW - Dimerization

KW - Electron transfer

KW - Enzymology

KW - Fatty acids

KW - Oxygenase

KW - BM3

KW - flavocytochrome P450 BM3 from Bacillus megaterium

KW - CO

KW - Carbon monoxide

KW - AUC

KW - analytical ultracentrifugation

KW - eT

KW - NOS

KW - Nitric oxide synthase

UR - http://www.scopus.com/inward/record.url?scp=26844498754&partnerID=8YFLogxK

U2 - 10.1016/j.febslet.2005.09.023

DO - 10.1016/j.febslet.2005.09.023

M3 - Article

C2 - 16214136

AN - SCOPUS:26844498754

VL - 579

SP - 5582

EP - 5588

JO - FEBS Letters

JF - FEBS Letters

SN - 0014-5793

IS - 25

ER -

The dimeric form of flavocytochrome P450 BM3 is catalytically functional as a fatty acid hydroxylase

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