New oxyfunctionalization capabilities for .omega.-hydroxylases.  Asymmetric aliphatic sulfoxidation and branched ether demethylation

Katopodis, Andreas; Smith, Homer A.; May, Sheldon W.

doi:10.1021/ja00211a033

Cited by 62 publications

(19 citation statements)

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“…In addition to the energetically demanding hydroxylation of unactivated aliphatic methyl groups, AlkB has been shown to catalyze a number of other characteristic reactions (7)(8)(9). These reactions are consistent with the involvement of high-valent, electrophilic catalytic intermediates comparable to those found in cytochrome P450 (10) and a number of other nonheme iron proteins (11).…”

supporting

confidence: 53%

Mössbauer studies of alkane ω-hydroxylase: Evidence for a diiron cluster in an integral-membrane enzyme

Shanklin

Achim

Schmidt

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

203

160

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The gene encoding the alkane -hydroxylase (AlkB; EC 1.14.15.3) from Pseudomonas oleovorans was expressed in Escherichia coli. The integral-membrane protein was purified as nearly homogeneous protein vesicles by differential ultracentrifugation and HPLC cation exchange chromatography without the detergent solubilization normally required for membrane proteins. Purified AlkB had specific activity of up to 5 units͞mg for octane-dependent NADPH consumption. Mössbauer studies of AlkB showed that it contains an exchange-coupled dinuclear iron cluster of the type found in soluble diiron proteins such as hemerythrin, ribonucleotide reductase, methane monooxygenase, stearoyl-acyl carrier protein (ACP) ⌬ 9 desaturase, rubrerythrin, and purple acid phosphatase. In the as-isolated enzyme, the cluster contains an antiferromagnetically coupled pair of high-spin Fe(III) sites, with an occupancy of up to 0.9 cluster per AlkB. The diferric cluster could be reduced by sodium dithionite, and the diferrous state was found to be stable in air. When both O 2 and substrate (octane) were added, however, the diferrous cluster was quantitatively reoxidized, proving that the diiron cluster occupies the active site. Mössbauer data on reduced AlkB are consistent with a cluster coordination rich in nitrogen-containing ligands. New sequence analyses indicate that at least 11 nonheme integral-membrane enzymes, including AlkB, contain the 8-histidine motif required for catalytic activity in stearoyl-CoA desaturase. Based on our Mössbauer studies of AlkB, we propose that the integral-membrane enzymes in this family contain diiron clusters. Because these enzymes catalyze a diverse range of oxygenation reactions, this proposal suggests a greatly expanded role for diiron clusters in O 2 -activation biochemistry.

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supporting

confidence: 53%

Mössbauer studies of alkane ω-hydroxylase: Evidence for a diiron cluster in an integral-membrane enzyme

Shanklin

Achim

Schmidt

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

203

160

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“…For example, the well-characterized alkane hydroxylase system in P. putida GPo1 is expressed by this organism after growth on C 6 to C 12 n-alkanes. While n-octane-grown cells of this organism have been reported not to oxidize MTBE (26), this alkane hydroxylase is known to O-dealkylate a variety of methoxylated alkane derivatives (15). Note also that a P. aeruginosa strain that is believed to express alkane hydroxylase activity after growth on C 5 to C 8 n-alkanes has also been reported to oxidize nbutane, even though this compound does not support cell growth (29).…”

Section: Cometabolism Of Mtbementioning

confidence: 99%

Cometabolism ofMethyl tertiary Butyl Ether and Gaseous n -Alkanes by Pseudomonas mendocina KR-1 Grown on C ₅ toC ₈ n -Alkanes

Smith

O’Reilly

Hyman

2003

Appl Environ Microbiol

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Pseudomonas mendocina KR-1 grew well on toluene, n-alkanes (C 5 to C 8 ), and 1°alcohols (C 2 to C 8 ) but not on other aromatics, gaseous n-alkanes (C 1 to C 4 ), isoalkanes (C 4 to C 6 ), 2°alcohols (C 3 to C 8 ), methyl tertiary butyl ether (MTBE), or tertiary butyl alcohol (TBA). Cells grown under carbon-limited conditions on n-alkanes in the presence of MTBE (42 mol) oxidized up to 94% of the added MTBE to TBA. Less than 3% of the added MTBE was oxidized to TBA when cells were grown on either 1°alcohols, toluene, or dextrose in the presence of MTBE. Concentrated n-pentane-grown cells oxidized MTBE to TBA without a lag phase and without generating tertiary butyl formate (TBF) as an intermediate. Neither TBF nor TBA was consumed by n-pentanegrown cells, while formaldehyde, the expected C 1 product of MTBE dealkylation, was rapidly consumed. Similar K s values for MTBE were observed for cells grown on C 5 to C 8 n-alkanes (12.95 ؎ 2.04 mM), suggesting that the same enzyme oxidizes MTBE in cells grown on each n-alkane. All growth-supporting n-alkanes (C 5 to C 8 ) inhibited MTBE oxidation by resting n-pentane-grown cells. Propane (K i ‫؍‬ 53 M) and n-butane (K i ‫؍‬ 16 M) also inhibited MTBE oxidation, and both gases were also consumed by cells during growth on n-pentane. Cultures grown on C 5 to C 8 n-alkanes also exhibited up to twofold-higher levels of growth in the presence of propane or n-butane, whereas no growth stimulation was observed with methane, ethane, MTBE, TBA, or formaldehyde. The results are discussed in terms of their impacts on our understanding of MTBE biodegradation and cometabolism.

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“…It is a three-component system comprising: (i) a soluble NADH-rubredoxin reductase [2], (ii) a soluble rubredoxin [3] and (iii) the integral membrane oxygenase AlkB (EC 1.14.15.3) [4^6]. In addition to the energetically demanding hydroxylation of inactivated methyl groups, AlkB has been shown to catalyze other reactions such as epoxidation of alkenes [7,8]. Reactions of inactivated hydrocarbons are reminiscent of transformations involving high-valent catalytic intermediates mediated by cytochrome P450 and methane monooxygenase [9^11].…”

Section: Introductionmentioning

confidence: 99%

Evidence linking the Pseudomonas oleovorans alkane ω‐hydroxylase, an integral membrane diiron enzyme, and the fatty acid desaturase family

Shanklin

Whittle

2003

FEBS Letters

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Pseudomonas oleovorans alkane g g-hydroxylase (AlkB) is an integral membrane diiron enzyme that shares a requirement for iron and oxygen for activity in a manner similar to that of the non-heme integral membrane desaturases, epoxidases, acetylenases, conjugases, ketolases, decarbonylase and methyl oxidases. No overall sequence similarity is detected between AlkB and these desaturase-like enzymes by computer algorithms; however, they do contain a series of histidine residues in a similar relative positioning with respect to hydrophobic regions thought to be transmembrane domains. To test whether these conserved histidine residues are functionally equivalent to those of the desaturase-like enzymes we used scanning alanine mutagenesis to test if they are essential for activity of AlkB. These experiments show that alanine substitution of any of the eight conserved histidines results in complete inactivation, whereas replacement of three non-conserved histidines in close proximity to the conserved residues, results in only partial inactivation. These data provide the ¢rst experimental support for the hypotheses: (i) that the histidine motif in AlkB is equivalent to that in the desaturase-like enzymes and (ii) that the conserved histidine residues play a vital role such as coordinating the Fe ions comprising the diiron active site.

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New oxyfunctionalization capabilities for .omega.-hydroxylases. Asymmetric aliphatic sulfoxidation and branched ether demethylation

Cited by 62 publications

References 2 publications

Mössbauer studies of alkane ω-hydroxylase: Evidence for a diiron cluster in an integral-membrane enzyme

Mössbauer studies of alkane ω-hydroxylase: Evidence for a diiron cluster in an integral-membrane enzyme

Cometabolism ofMethyl tertiary Butyl Ether and Gaseous n -Alkanes by Pseudomonas mendocina KR-1 Grown on C ₅ toC ₈ n -Alkanes

Evidence linking the Pseudomonas oleovorans alkane ω‐hydroxylase, an integral membrane diiron enzyme, and the fatty acid desaturase family

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New oxyfunctionalization capabilities for .omega.-hydroxylases. Asymmetric aliphatic sulfoxidation and branched ether demethylation

Cited by 62 publications

References 2 publications

Mössbauer studies of alkane ω-hydroxylase: Evidence for a diiron cluster in an integral-membrane enzyme

Mössbauer studies of alkane ω-hydroxylase: Evidence for a diiron cluster in an integral-membrane enzyme

Cometabolism ofMethyl tertiary Butyl Ether and Gaseous n -Alkanes by Pseudomonas mendocina KR-1 Grown on C 5 toC 8 n -Alkanes

Evidence linking the Pseudomonas oleovorans alkane ω‐hydroxylase, an integral membrane diiron enzyme, and the fatty acid desaturase family

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Product

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Cometabolism ofMethyl tertiary Butyl Ether and Gaseous n -Alkanes by Pseudomonas mendocina KR-1 Grown on C ₅ toC ₈ n -Alkanes