Molecular analysis of the soluble butane monooxygenase from 'Pseudomonas butanovora'.

'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 sigma(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.

Biochemical, molecular, and genetic analyses of the acetone carboxylases from Xanthobacter autotrophicus strain Py2 and Rhodobacter capsulatus strain B10.

Acetone carboxylase is the key enzyme of bacterial acetone metabolism, catalyzing the condensation of acetone and CO(2) to form acetoacetate. In this study, the acetone carboxylase of the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus was purified to homogeneity and compared to that of Xanthobacter autotrophicus strain Py2, the only other organism from which an acetone carboxylase has been purified. The biochemical properties of the enzymes were virtually indistinguishable, with identical subunit compositions (alpha(2)beta(2)gamma(2) multimers of 85-, 78-, and 20-kDa subunits), reaction stoichiometries (CH(3)COCH(3) + CO(2) + ATP-->CH(3)COCH(2)COO(-) + H(+) + AMP + 2P(i)), and kinetic properties (K(m) for acetone, 8 microM; k(cat) = 45 min(-1)). Both enzymes were expressed to high levels (17 to 25% of soluble protein) in cells grown with acetone as the carbon source but were not present at detectable levels in cells grown with other carbon sources. The genes encoding the acetone carboxylase subunits were identified by transposon mutagenesis of X. autotrophicus and sequence analysis of the R. capsulatus genome and were found to be clustered in similar operons consisting of the genes acxA (beta subunit), acxB (alpha subunit), and acxC (gamma subunit). Transposon mutagenesis of X. autotrophicus revealed a requirement of sigma(54) and a sigma(54)-dependent transcriptional activator (AcxR) for acetone-dependent growth and acetone carboxylase gene expression. A potential sigma(54)-dependent promoter 122 bp upstream of X. autotrophicus acxABC was identified. An AcxR gene homolog was identified 127 bp upstream of acxA in R. capsulatus, but this activator lacked key features of sigma(54)-dependent activators, and the associated acxABC lacked an apparent sigma(54)-dependent promoter, suggesting that sigma(54) is not required for expression of acxABC in R. capsulatus. These studies reveal a conserved strategy of ATP-dependent acetone carboxylation and the involvement of transcriptional enhancers in acetone carboxylase gene expression in gram-negative acetone-utilizing bacteria.