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Molecular analysis of the soluble butane monooxygenase from ‘Pseudomonas butanovora’^a

Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA¹

Author for correspondence: Daniel J. Arp. Tel: +1 541 737 1294. Fax: +1 541 737 5310. e-mail: arpd{at}bcc.orst.edu

‘Pseudomonas butanovora’ is capable of growth with butane via the oxidation of butane to 1-butanol, which is catalysed by a soluble butane monooxygenase (sBMO). In vitro oxidation of ethylene (an alternative substrate for sBMO) was reconstituted in the soluble portion of cell extracts and was NADH-dependent. Butane monooxygenase was separated into three components which were obligately required for substrate oxidation. The N-terminal sequences of the peptides associated with butane monooxygenase led to the cloning and sequencing of the 5797 nucleotide bmo gene cluster. Comparisons of the deduced amino acid sequences with other multicomponent monooxygenases suggest that sBMO is a multimeric hydroxylase with 61, 45 and 19 kDa subunits encoded by bmoXYZ, a 40 kDa oxidoreductase encoded by bmoC, and a 15 kDa regulatory protein encoded by bmoB. A sixth structural gene (bmoD) encodes a 9·6 kDa protein with similarity exclusively to mmoD (orfY), a putative metal centre assembly protein of the soluble methane monooxygenases. Insertional inactivation of bmoX resulted in a mutant ‘P. butanovora’ strain incapable of growth with butane. A putative promoter element characteristic of promoters associated with ⁵⁴-dependent transcription initiation was located upstream of the bmo genes. Expression of all six genes was detected in butane-induced cells. Butane monooxygenase from ‘P. butanovora’ aligns most closely with non-haem carboxylate-bridged diiron monooxygenases and, moreover, contains the characteristic iron-binding motif. The structural and mechanistic implications of the high sequence identity (up to 64%) between the peptides of butane monooxygenase and methane monooxygenases are discussed.

Keywords: butane metabolism, alkane oxidation, diiron monooxygenase

Abbreviations: BMOB, butane monooxygenase regulatory protein; BMOD, bmoD gene product; BMOH, butane monooxygenase hydroxylase; BMOR, butane monooxygenase reductase; MMOB, methane monooxygenase regulatory protein; MMOD, mmoD (orfY) gene product; MMOH, methane monooxygenase hydroxylase; MMOR, methane monooxygenase reductase; pMMO, particulate methane monooxygenase; sBMO, soluble butane monooxygenase; sMMO, soluble methane monooxygenase

^a The GenBank accession number for the bmoXYBZDC sequence is AY093933.

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