Biochemical, molecular, and genetic analyses of the acetone carboxylases from Xanthobacter autotrophicus strain Py2 and Rhodobacter capsulatus strain B10
Biochemical, molecular, and genetic analyses of the acetone carboxylases from Xanthobacter autotrophicus strain Py2 and Rhodobacter capsulatus strain B10
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DOI：
10.1128/jb.184.11.2969-2977.2002
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发表时间：
2002-06-01
影响因子：
3.2
通讯作者：
Ensign, SA
中科院分区：
文献类型：
--
作者：
Sluis, MK;Larsen, RA;Ensign, SA
丙酮羧基酶是细菌丙酮代谢的关键酶，催化丙酮和二氧化碳缩合生成乙酰乙酸酯。在这项研究中，紫色非硫光合细菌胶囊红假单胞菌的丙酮羧基酶被纯化为均一，并与自养黄杆菌菌株PY2的丙酮羧基酶进行比较，黄杆菌自养菌株PY2是唯一纯化丙酮羧基酶的另一种生物。这些酶的生化性质几乎无法区分，具有相同的亚基组成(α(2)β(2)伽马(2))、反应化学计量(CH3COCH3+CO2+ATP-->；CH3COCH2COO-+H++AMP+2P(I))和动力学性质(丙酮的K-m，8微米；k(CAT)=45min(-1))。在以丙酮为碳源生长的细胞中，这两种酶的表达水平都很高(可溶蛋白质的17%至25%)，而在以其他碳源生长的细胞中，这两种酶的表达水平都不是很高。通过转座子诱变和基因序列分析，发现编码丙酮羧基酶亚基的基因由acxA(β亚基)、acxB(α亚基)和acxC(伽马亚基)组成。转座子诱变发现自养X.Sigma(54)和依赖于Sigma(54)的转录激活因子(AcxR)对丙酮依赖生长和丙酮羧基酶基因表达是必需的。在自养X.acxABC上游的122个碱基上发现了一个潜在的依赖于Sigma(54)的启动子。在胶囊乳杆菌acxA上游127bp处发现了一个AcxR基因同源基因，但该激活子缺乏依赖sigma(54)的激活子的关键特征，相关的acxABC也缺乏明显的依赖sigma(54)的启动子，这表明acxABC的表达不需要sigma(54)。这些研究揭示了一种依赖于ATP的丙酮羧化的保守策略，以及转录增强子参与了革兰氏阴性丙酮利用细菌丙酮羧基酶基因的表达。
Acetone carboxylase is the key enzyme of bacterial acetone metabolism, catalyzing the condensation of acetone and CO2 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 (CH3COCH3 + CO2 + ATP-->CH3COCH2COO- + H+ + AMP + 2P(i)), and kinetic properties (K-m for acetone, 8 muM; 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.