Zhang Daohai, Li Nan, Lok Shee-Mei, Zhang Lian-Hui, Swaminathan Kunchithapadam
Department of Pathology, National University of Singapore, 5 Lower Kent Ridge Road, Singapore 119074.
J Biol Chem. 2003 Sep 12;278(37):35428-34. doi: 10.1074/jbc.M302616200. Epub 2003 Jun 20.
Isomaltulose synthase from Klebsiella sp. LX3 (PalI, EC 5.4.99.11) catalyzes the isomerization of sucrose to produce isomaltulose (alpha-D-glucosylpyranosyl-1,6-D-fructofuranose) and trehalulose (alpha-D-glucosylpyranosyl-1,1-d-fructofuranose). The PalI structure, solved at 2.2-A resolution with an R-factor of 19.4% and Rfree of 24.2%, consists of three domains: an N-terminal catalytic (beta/alpha)8 domain, a subdomain between N beta 3 and N alpha 3, and a C-terminal domain having seven beta-strands. The active site architecture of PalI is identical to that of other glycoside hydrolase family 13 members, suggesting a similar mechanism in substrate binding and hydrolysis. However, a unique RLDRD motif in the proximity of the active site has been identified and shown biochemically to be responsible for sucrose isomerization. A two-step reaction mechanism for hydrolysis and isomerization, which occurs in the same pocket is proposed based on both the structural and biochemical data. Selected C-terminal truncations have been shown to reduce and even abolish the enzyme activity, consistent with the predicted role of the C-terminal residues in the maintenance of enzyme conformation and active site topology.
来自克雷伯氏菌属LX3的异麦芽糖合酶（PalI，EC 5.4.99.11）催化蔗糖异构化生成异麦芽糖（α-D-吡喃葡萄糖基-1,6-D-呋喃果糖）和海藻糖（α-D-吡喃葡萄糖基-1,1-D-呋喃果糖）。PalI的结构在2.2 Å分辨率下解析得到，R因子为19.4%，Rfree为24.2%，由三个结构域组成：一个N端催化（β/α）8结构域、Nβ3和Nα3之间的一个亚结构域以及一个具有七条β链的C端结构域。PalI的活性位点结构与其他糖苷水解酶家族13成员的相同，表明在底物结合和水解方面有类似机制。然而，已在活性位点附近鉴定出一个独特的RLDRD基序，并通过生化实验证明其负责蔗糖异构化。基于结构和生化数据，提出了在同一口袋中发生的水解和异构化的两步反应机制。已证明选定的C端截短会降低甚至消除酶活性，这与C端残基在维持酶构象和活性位点拓扑结构中的预测作用一致。
