Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität, Münster, Germany.
J Appl Microbiol. 2010 Jul;109(1):79-90. doi: 10.1111/j.1365-2672.2009.04631.x. Epub 2009 Nov 19.
Detailed knowledge about the enzymes responsible for conversion of C(3) and C(4) compounds will be helpful to establish the bacterial strain Ralstonia eutropha as platform for the production of biotechnologically interesting compounds. Although various studies about these enzymes were accomplished in the past, some contradicting information about the enzyme pattern in this bacterium still exists. To resolve these discrepancies, the C(3) /C(4) metabolism was reinvestigated after the genome sequence of this bacterium became available.
In silico analysis of genome sequence revealed putative genes coding for NAD(P)(+) -dependent malic enzymes (Mae), phoshoenolpyruvate carboxykinase (Pck), phosphoenolpyruvate carboxylase (Ppc), phosphoenolpyruvate synthase (Pps) and pyruvate carboxylase (Pyc). Reverse transcription PCR revealed constitutive expression of mae and pck genes, whereas no transcripts of pyc and ppc were found. Expression of active NADP(+) -dependent MaeB and Pck and absence of Pyc and Ppc was confirmed by spectrophotometric enzyme assays.
The data reported in this study suggest that two enzymes, (i) MaeB and (ii) Pck, mediate between the C(3) and C(4) intermediates in R. eutropha H16. The enzymatic conversion of pyruvate into phosphoenolpyruvate (PEP) is catalysed by Pps, and an NADH(+) -dependent Mdh mediates the reversible conversion of malate and oxaloacetate.
An increased knowledge of the enzymes mediating between C(3) and C(4) intermediates in R. eutropha will facilitate metabolic engineering.
详细了解负责 C(3)和 C(4)化合物转化的酶，将有助于将细菌菌株 Ralstonia eutropha 建立为生产生物技术感兴趣的化合物的平台。尽管过去已经完成了各种关于这些酶的研究，但关于该细菌中酶模式的一些相互矛盾的信息仍然存在。为了解决这些差异，在该细菌的基因组序列可用后，重新研究了 C(3)/C(4)代谢。
对基因组序列的计算机分析揭示了编码 NAD(P)(+) 依赖性苹果酸酶 (Mae)、磷酸烯醇丙酮酸羧激酶 (Pck)、磷酸烯醇丙酮酸羧化酶 (Ppc)、磷酸烯醇丙酮酸合酶 (Pps) 和丙酮酸羧化酶 (Pyc) 的推定基因。逆转录 PCR 显示 mae 和 pck 基因的组成型表达，而没有发现 pyc 和 ppc 的转录物。通过分光光度酶测定证实了活性的 NADP(+) 依赖性 MaeB 和 Pck 的表达以及 Pyc 和 Ppc 的不存在。
本研究报告的数据表明，两种酶 (i) MaeB 和 (ii) Pck，在 R. eutropha H16 中介导 C(3)和 C(4)中间体之间的转化。丙酮酸转化为磷酸烯醇丙酮酸 (PEP) 的酶促反应由 Pps 催化，NADH(+) 依赖性 Mdh 介导苹果酸和草酰乙酸的可逆转化。
增加对 R. eutropha 中介导 C(3)和 C(4)中间体之间的酶的了解将有助于代谢工程。
