Reddick Jason J, Sirkisoon Sherona, Dahal Rejwi Acharya, Hardesty Grant, Hage Natalie E, Booth William T, Quattlebaum Amy L, Mills Suzette N, Meadows Victoria G, Adams Sydney L H, Doyle Jennifer S, Kiel Brittany E
Department of Chemistry and Biochemistry, University of North Carolina at Greensboro , Greensboro, North Carolina 27402, United States.
Biochemistry. 2017 Oct 24;56(42):5698-5711. doi: 10.1021/acs.biochem.7b00778. Epub 2017 Oct 6.
The genome of Bacillus subtilis strain 168 contains the mother cell metabolic gene (mmg) operon that encodes homologues from the methylcitric acid cycle. We showed that the three genes, mmgDE and yqiQ(mmgF), provide three of the five steps of the methylcitric acid cycle. We also showed that the fourth step can be supplied by citB (aconitase), and we suggest that the fifth missing step, the propionyl-CoA synthetase, is probably skipped because the β-oxidation of methyl-branched fatty acids by the enzymes encoded by mmgABC should produce propionyl-CoA. We also noted interesting enzymology for MmgD and MmgE. First, MmgD is a bifunctional citrate synthase/2-methylcitrate synthase with 2.3-fold higher activity as a 2-methylcitrate synthase. This enzyme catalyzes the formation of either (2S,3R)- or (2R,3S)-2-methylcitrate, but reports of 2-methylcitrate synthases from other species indicated that they produced the (2S,3S) isomer. However, we showed that MmgD and PrpC (from Escherichia coli) in fact produce the same stereoisomer. Second, the MmgE enzyme is not a stereospecific 2-methylcitrate dehydratase because it can dehydrate at least two of the four diastereomers of 2-methylcitrate to yield either (E)-2-methylaconitate or (Z)-2-methylaconitate. We also showed for the first time that the E. coli homologue PrpD exhibited the same lack of stereospecificity. However, the physiological pathways proceed via (Z)-2-methylaconitate, which served as the substrate for the citB enzyme in the synthesis of 2-methylisocitrate. We completed our characterization of this pathway by showing that the 2-methylisocitrate produced by CitB is converted to pyruvate and succinate by the enzyme YqiQ(MmgF).
枯草芽孢杆菌168菌株的基因组包含母细胞代谢基因（mmg）操纵子，该操纵子编码甲基柠檬酸循环中的同源物。我们发现，mmgDE和yqiQ（mmgF）这三个基因提供了甲基柠檬酸循环五个步骤中的三个。我们还发现第四步可由citB（乌头酸酶）提供，并且我们认为缺失的第五步，即丙酰辅酶A合成酶，可能被跳过了，因为mmgABC编码的酶对甲基支链脂肪酸的β氧化应该会产生丙酰辅酶A。我们还注意到了MmgD和MmgE有趣的酶学性质。首先，MmgD是一种双功能柠檬酸合酶/2-甲基柠檬酸合酶，作为2-甲基柠檬酸合酶时活性高2.3倍。这种酶催化形成（2S,3R）-或（2R,3S）-2-甲基柠檬酸，但其他物种的2-甲基柠檬酸合酶的报道表明它们产生的是（2S,3S）异构体。然而，我们发现MmgD和PrpC（来自大肠杆菌）实际上产生的是相同的立体异构体。其次，MmgE酶不是立体特异性的2-甲基柠檬酸脱水酶，因为它可以使2-甲基柠檬酸的四种非对映异构体中的至少两种脱水，生成（E）-2-甲基乌头酸或（Z）-2-甲基乌头酸。我们还首次发现大肠杆菌同源物PrpD也表现出同样缺乏立体特异性的情况。然而，生理途径是通过（Z）-2-甲基乌头酸进行的，它作为citB酶合成2-甲基异柠檬酸的底物。我们通过证明CitB产生的2-甲基异柠檬酸被YqiQ（MmgF）酶转化为丙酮酸和琥珀酸，完成了对该途径的表征。
