Bandell M, Ansanay V, Rachidi N, Dequin S, Lolkema J S
Department of Microbiology, Groningen Biotechnology and Biomolecular Sciences Institute, University of Groningen, 9751NN Haren, The Netherlands.
J Biol Chem. 1997 Jul 18;272(29):18140-6. doi: 10.1074/jbc.272.29.18140.
Membrane potential generation via malate/lactate exchange catalyzed by the malate carrier (MleP) of Lactococcus lactis, together with the generation of a pH gradient via decarboxylation of malate to lactate in the cytoplasm, is a typical example of a secondary proton motive force-generating system. The mleP gene was cloned, sequenced, and expressed in a malolactic fermentation-deficient L. lactis strain. Functional analysis revealed the same properties as observed in membrane vesicles of a malolactic fermentation-positive strain. MleP belongs to a family of secondary transporters in which the citrate carriers from Leuconostoc mesenteroides (CitP) and Klebsiella pneumoniae (CitS) are found also. CitP, but not CitS, is also involved in membrane potential generation via electrogenic citrate/lactate exchange. MleP, CitP, and CitS were analyzed for their substrate specificity. The 2-hydroxycarboxylate motif R1R2COHCOOH, common to the physiological substrates, was found to be essential for transport although some 2-oxocarboxylates could be transported to a lesser extent. Clear differences in substrate specificity among the transporters were observed because of different tolerances toward the R substituents at the C2 atom. Both MleP and CitP transport a broad range of 2-hydroxycarboxylates with R substituents ranging in size from two hydrogen atoms (glycolate) to acetyl and methyl groups (citromalate) for MleP and two acetyl groups (citrate) for CitP. CitS was much less tolerant and transported only citrate and at a low rate citromalate. The substrate specificities are discussed in the context of the physiological function of the transporters.
乳酸乳球菌的苹果酸载体（MleP）催化苹果酸/乳酸交换产生膜电位，同时细胞质中苹果酸脱羧生成乳酸产生pH梯度，这是次级质子动力产生系统的典型例子。mleP基因被克隆、测序并在缺乏苹果酸-乳酸发酵的乳酸乳球菌菌株中表达。功能分析显示其具有与苹果酸-乳酸发酵阳性菌株的膜囊泡中观察到的相同特性。MleP属于次级转运蛋白家族，其中还发现了来自肠系膜明串珠菌（CitP）和肺炎克雷伯菌（CitS）的柠檬酸载体。CitP，但不是CitS，也通过电中性柠檬酸/乳酸交换参与膜电位的产生。分析了MleP、CitP和CitS的底物特异性。发现生理底物共有的2-羟基羧酸盐基序R1R2COHCOOH对于转运至关重要，尽管一些2-氧代羧酸盐可以在较小程度上被转运。由于对C2原子上R取代基的耐受性不同，观察到转运蛋白之间底物特异性存在明显差异。MleP和CitP都转运范围广泛的2-羟基羧酸盐，MleP的R取代基大小从两个氢原子（乙醇酸）到乙酰基和甲基（柠苹酸），CitP的R取代基为两个乙酰基（柠檬酸）。CitS的耐受性要低得多，只转运柠檬酸，柠苹酸的转运速率也很低。在转运蛋白的生理功能背景下讨论了底物特异性。
