Biochemical, Molecular, and Genetic Analyses of the Acetone Carboxylases from Xanthobacter autotrophicus Strain Py2 and Rhodobacter capsulatus Strain B10

Abstract

ABSTRACT 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 (α 2 β 2 γ 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 μM; 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 (β subunit), acxB (α subunit), and acxC (γ subunit). Transposon mutagenesis of X. autotrophicus revealed a requirement of σ 54 and a σ 54 -dependent transcriptional activator (AcxR) for acetone-dependent growth and acetone carboxylase gene expression. A potential σ 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 σ 54 -dependent activators, and the associated acxABC lacked an apparent σ 54 -dependent promoter, suggesting that σ 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.