Hallis T M, Lei Y, Que N L, Liu H
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Biochemistry. 1998 Apr 7;37(14):4935-45. doi: 10.1021/bi9725529.
The 3,6-dideoxyhexoses can be found in the cell wall lipopolysaccharide of Gram-negative bacteria, where they have been shown to be the dominant antigenic determinants. All naturally occurring 3,6-dideoxyhexoses, with colitose as the only exception, are biosynthesized via a complex pathway that begins with CDP-d-glucose. Included in this pathway is CDP-paratose synthase, an essential enzyme in the formation of the 3,6-dideoxy sugars, CDP-paratose and CDP-tyvelose. Recently, the gene encoding CDP-paratose synthase in Salmonella typhi, rfbS, has been identified and sequenced [Verma, N., and Reeves, P. (1989) J. Bacteriol. 171, 5694-5701]. On the basis of this information, we have amplified the rfbS gene by polymerase chain reaction (PCR) from S. typhi and cloned this gene into a pET-24(+) vector. Expression and purification of CDP-paratose synthase have allowed us to fully characterize the catalytic properties of this enzyme, which is a homodimeric protein with a preference for NADPH over NADH. It catalyzes the stereospecific hydride transfer of the pro-S hydrogen from the C-4' position of the reduced coenzyme to C-4 of the substrate, CDP-3,6-dideoxy-D-glycero-D-glycero-4-hexulose. The overall equilibrium of this catalysis greatly favors the formation of the reduced sugar product and the oxidized coenzyme. Interestingly, this enzyme also exhibits a high affinity for NADPH with a much smaller dissociation constant (Kia) of 0.005 +/- 0.002 microM compared to the Km of 26 +/- 8 microM for NADPH. While this unusual property complicated the interpretation of the kinetic data, the kinetic mechanism of CDP-paratose synthase as explored by the combination of bisubstrate kinetic analysis, product inhibition studies, and dead-end competitive inhibition studies is most consistent with a Theorell-Chance mechanism. The present study on CDP-paratose synthase, a likely new member of the short-chain dehydrogenase family, represents the first detailed characterization of this type of ketohexose reductase, many of which may share similar properties with CDP-paratose synthase.
3,6 -二脱氧己糖可存在于革兰氏阴性菌的细胞壁脂多糖中，在那里它们已被证明是主要的抗原决定簇。除了可洛糖外，所有天然存在的3,6 -二脱氧己糖都是通过一条从CDP - D -葡萄糖开始的复杂途径生物合成的。该途径包括CDP - 副糖合成酶，它是3,6 -二脱氧糖、CDP - 副糖和CDP - 泰威糖形成过程中的一种必需酶。最近，伤寒沙门氏菌中编码CDP - 副糖合成酶的基因rfbS已被鉴定并测序[Verma, N., and Reeves, P. (1989) J. Bacteriol. 171, 5694 - 5701]。基于这一信息，我们通过聚合酶链反应（PCR）从伤寒沙门氏菌中扩增了rfbS基因，并将该基因克隆到pET - 24(+)载体中。CDP - 副糖合成酶的表达和纯化使我们能够全面表征该酶的催化特性，它是一种同源二聚体蛋白，对NADPH的偏好高于NADH。它催化还原型辅酶C - 4'位的前手性氢向底物CDP - 3,6 -二脱氧 - D -甘油 - D -甘油 - 4 -己酮糖的C - 4进行立体专一性氢转移。该催化反应的总体平衡极大地有利于还原糖产物和氧化型辅酶的形成。有趣的是，该酶对NADPH也表现出高亲和力，其解离常数（Kia）为0.005±0.002 microM，远小于NADPH的Km值26±8 microM。虽然这种不寻常的性质使动力学数据的解释变得复杂，但通过双底物动力学分析、产物抑制研究和终产物竞争性抑制研究相结合所探索的CDP - 副糖合成酶的动力学机制与Theorell - Chance机制最为一致。目前对CDP - 副糖合成酶（短链脱氢酶家族的一个可能新成员）的研究代表了对这类酮己糖还原酶的首次详细表征，其中许多可能与CDP - 副糖合成酶具有相似的性质。
