Flavocytochrome P450 BM3 mutant W1046A is a NADH-dependent fatty acid hydroxylase: Implications for the mechanism of electron transfer in the P450 BM3 dimer

2011 ◽  
Vol 507 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Hazel M. Girvan ◽  
Adrian J. Dunford ◽  
Rajasekhar Neeli ◽  
Idorenyin S. Ekanem ◽  
Timothy N. Waltham ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Fatty Acid ◽  
P450 Bm3 ◽  
Fatty Acid Hydroxylase

scholarly journals Roles of key active-site residues in flavocytochrome P450 BM3

1999 ◽  
Vol 339 (2) ◽  
pp. 371-379 ◽  
Author(s):  
Michael A. NOBLE ◽  
Caroline S. MILES ◽  
Stephen K. CHAPMAN ◽  
Dominikus A. LYSEK ◽  
Angela C. MACKAY ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Active Site ◽  
Side Chain ◽  
Acid Oxidation ◽  
Dodecanoic Acid ◽  
Wild Type ◽  
Active Site Residues ◽  
P450 Bm3

The effects of mutation of key active-site residues (Arg-47, Tyr-51, Phe-42 and Phe-87) in Bacillus megaterium flavocytochrome P450 BM3 were investigated. Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate: R47A mutant, Km 859 µM, kcat 3960 min-1; Y51F mutant, Km 432 µM, kcat 6140 min-1; wild-type, Km 288 µM, kcat 5140 min-1). A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (ΔG‡) resulting from a smaller ΔG of substrate binding. The side chain of Phe-42 acts as a phenyl ‘cap ’ over the mouth of the substrate-binding channel. With mutant F42A, Km is massively increased and kcat is decreased for oxidation of both laurate (Km 2.08 mM, kcat 2450 min-1) and arachidonate (Km 34.9 µM, kcat 14620 min-1; compared with values of 4.7 µM and 17100 min-1 respectively for wild-type). Amino acid Phe-87 is critical for efficient catalysis. Mutants F87G and F87Y not only exhibit increased Km and decreased kcat values for fatty acid oxidation, but also undergo an irreversible conversion process from a ‘fast ’ to a ‘slow ’ rate of substrate turnover [for F87G (F87Y)-catalysed laurate oxidation: kcat ‘fast ’, 760 (1620) min-1; kcat ‘slow ’, 48.0 (44.6) min-1; kconv (rate of conversion from fast to slow form), 4.9 (23.8) min-1]. All mutants showed less than 10% uncoupling of NADPH oxidation from fatty acid oxidation. The rate of FMN-to-haem electron transfer was shown to become rate-limiting in all mutants analysed. For wild-type P450 BM3, the rate of FMN-to-haem electron transfer (8340 min-1) is twice the steady-state rate of oxidation (4100 min-1), indicating that other steps contribute to rate limitation. Active-site structures of the mutants were probed with the inhibitors 12-(imidazolyl)dodecanoic acid and 1-phenylimidazole. Mutant F87G binds 1-phenylimidazole > 10-fold more tightly than does the wild-type, whereas mutant Y51F binds the haem-co-ordinating fatty acid analogue 12-(imidazolyl)dodecanoic acid > 30-fold more tightly than wild-type.

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

FEBS Letters ◽  
2005 ◽  
Vol 579 (25) ◽  
pp. 5582-5588 ◽  
Author(s):  
Rajasekhar Neeli ◽  
Hazel M. Girvan ◽  
Andrew Lawrence ◽  
Martin J. Warren ◽  
David Leys ◽  
...  
Keyword(s):  
Fatty Acid ◽  
P450 Bm3 ◽  
Fatty Acid Hydroxylase ◽  
Dimeric Form

Novel biallelic FA2H mutations in a Japanese boy with fatty acid hydroxylase-associated neurodegeneration

2020 ◽  
Vol 42 (2) ◽  
pp. 217-221
Author(s):  
Masahiro Kawaguchi ◽  
Takayuki Sassa ◽  
Hiroyuki Kidokoro ◽  
Tomohiko Nakata ◽  
Kohji Kato ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Hydroxylase

scholarly journals LeTetR Positively Regulates 3-Hydroxylation of the Antifungal HSAF and Its Analogs in Lysobacter enzymogenes OH11

Molecules ◽  
2020 ◽  
Vol 25 (10) ◽  
pp. 2286
Author(s):  
Lingjun Yu ◽  
Vimmy Khetrapal ◽  
Fengquan Liu ◽  
Liangcheng Du
Keyword(s):  
Fatty Acid ◽  
Antifungal Activity ◽  
Biocontrol Agent ◽  
Biosynthetic Gene Cluster ◽  
E Coli ◽  
Lysobacter Enzymogenes ◽  
Heat Stable ◽  
Fatty Acid Hydroxylase ◽  
Hydroxylase Gene ◽  
Antibiotic Compounds

The biocontrol agent Lysobacter enzymogenes OH11 produces several structurally distinct antibiotic compounds, including the antifungal HSAF (Heat Stable Antifungal Factor) and alteramides, along with their 3-dehydroxyl precursors (3-deOH). We previously showed that the 3-hydroxylation is the final step of the biosynthesis and is also a key structural moiety for the antifungal activity. However, the procedure through which OH11 regulates the 3-hydroxylation is still not clear. In OH11, the gene orf3232 was predicted to encode a TetR regulator (LeTetR) with unknown function. Here, we deleted orf3232 and found that the LeTetR mutant produced very little HSAF and alteramides, while the 3-deOH compounds were not significantly affected. The production of HSAF and alteramides was restored in orf3232-complemented mutant. qRT-PCR showed that the deletion of orf3232 impaired the transcription of a putative fatty acid hydroxylase gene, orf2195, but did not directly affect the expression of the HSAF biosynthetic gene cluster (hsaf). When an enzyme extract from E. coli expressing the fatty acid hydroxylase gene, hsaf-orf7, was added to the LeTetR mutant, the production of HSAF and alteramides increased by 13–14 fold. This study revealed a rare function of the TetR family regulator, which positively controls the final step of the antifungal biosynthesis and thus controls the antifungal activity of the biocontrol agent.

Electron transfer in a P450 BM3/cytochrome b5 complex

1999 ◽  
Vol 27 (3) ◽  
pp. A108-A108
Author(s):  
Michael A. Noble ◽  
W.B. Ost Tobias ◽  
Caroline S. Miles ◽  
Laura Robledo ◽  
Stephen K. Chapman ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Cytochrome B5 ◽  
P450 Bm3

scholarly journals The CYPome of Sorangium cellulosum So ce56 and Identification of CYP109D1 as a New Fatty Acid Hydroxylase

2010 ◽  
Vol 17 (12) ◽  
pp. 1295-1305 ◽  
Author(s):  
Yogan Khatri ◽  
Frank Hannemann ◽  
Kerstin M. Ewen ◽  
Dominik Pistorius ◽  
Olena Perlova ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Sorangium Cellulosum ◽  
Fatty Acid Hydroxylase ◽  
Sorangium Cellulosum So Ce56

Role of Leu188 in the Fatty Acid Hydroxylase Activity of CYP102A1 from Bacillus megaterium

2016 ◽  
Vol 133 ◽  
pp. 35-42 ◽  
Author(s):  
Hyun-Hee Jang ◽  
Sun-Mi Shin ◽  
Sang Hoon Ma ◽  
Ga-Young Lee ◽  
Young Hee Joung ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Bacillus Megaterium ◽  
Hydroxylase Activity ◽  
Fatty Acid Hydroxylase

Cloning and Functional Characterization of CYP94A2, a Medium Chain Fatty Acid Hydroxylase from Vicia sativa

1999 ◽  
Vol 261 (1) ◽  
pp. 156-162 ◽  
Author(s):  
Renaud Le Bouquin ◽  
Franck Pinot ◽  
Irène Benveniste ◽  
Jean-Pierre Salaün ◽  
Francis Durst
Keyword(s):  
Fatty Acid ◽  
Functional Characterization ◽  
Chain Fatty Acid ◽  
Medium Chain Fatty Acid ◽  
Vicia Sativa ◽  
Medium Chain ◽  
Fatty Acid Hydroxylase
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