Membrane-bound, 2-keto-D-gluconate-yielding D-Gluconate dehydrogenase from "Gluconobacter dioxyacetonicus" IFO 3271:: Molecular properties and gene disruption
Membrane-bound, 2-keto-D-gluconate-yielding D-Gluconate dehydrogenase from "Gluconobacter dioxyacetonicus" IFO 3271:: Molecular properties and gene disruption
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DOI：
10.1128/aem.00493-07
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发表时间：
2007-10-01
影响因子：
4.4
通讯作者：
Matsushita, Kazunobu
中科院分区：
文献类型：
--
作者：
Toyama, Hirohide;Furuya, Naoko;Matsushita, Kazunobu
大多数葡萄糖酸菌在培养基中通过GA从D-葡萄糖同时产生和积累2-酮基-D-葡萄糖酸盐（2KGA）和5 KGA。2KGA由膜结合的含黄素腺嘌呤二核苷酸的GA 2-脱氢酶（FAD-GADH）产生。从“二氧丙酮葡萄糖杆菌”IFO 3271中纯化了FAD-GADH，并分析了三个亚基的N末端序列。根据N端序列设计PCR引物，克隆部分FAD-GADH基因作为PCR产物。使用该PCR产物，获得了含有完整FAD-GADH基因的基因片段，并最终确定了9，696 bp的核苷酸序列。克隆的序列有三个开放阅读框（ORF），gndS，gndL和gndC，分别对应于FAD-GADH的小，大和细胞色素c亚基。还发现了另外7个ORF，其中一个与β-δ-内酯酶相同，可能直接参与2KGA的产生。构建了gndL或sldA（负责5 KGA产生的基因）或两者都有缺陷的三种突变株。铁氰化物还原酶活性与GA在膜部分的gndL缺陷型菌株的野生型菌株的约60%下降，而在SLDA缺陷型菌株，活性与GA没有减少和活动与甘油，D-阿糖醇，和D-山梨醇消失。出乎意料的是，gndL和sldA缺陷的菌株（双突变体）仍然显示出GA活性。此外，在gndL和双突变株中仍观察到2KGA产生。在sldA和双突变菌株中根本没有观察到5 KGA产生。因此，看来“G。IFO 3271具有另一种与GA反应产生2KGA的膜结合酶。
Most Gluconobacter species produce and accumulate 2-keto-D-gluconate (2KGA) and 5KGA simultaneously from D-glucose via GA in culture medium. 2KGA is produced by membrane-bound flavin adenine dinucleotide-containing GA 2-dehydrogenase (FAD-GADH). FAD-GADH was purified from "Gluconobacter dioxyacetonicus" IFO 3271, and N-terminal sequences of the three subunits were analyzed. PCR primers were designed from the N-terminal sequences, and part of the FAD-GADH genes was cloned as a PCR product. Using this PCR product, gene fragments containing whole FAD-GADH genes were obtained, and finally the nucleotide sequence of 9,696 bp was determined. The cloned sequence had three open reading frames (ORFs), gndS, gndL, and gndC, corresponding to small, large, and cytochrome c subunits of FAD-GADH, respectively. Seven other ORFs were also found, one of which showed identity to glucono-delta-lactonase, which might be involved directly in 2KGA production. Three mutant strains defective in either gndL or sldA (the gene responsible for 5KGA production) or both were constructed. Ferricyanide-reductase activity with GA in the membrane fraction of the gndL-defective strain decreased by about 60% of that of the wild-type strain, while in the sldA-defective strain, activity with GA did not decrease and activities with glycerol, D-arabitol, and D-sorbitol disappeared. Unexpectedly, the strain defective in both gndL and sldA (double mutant) still showed activity with GA. Moreover, 2KGA production was still observed in gndL and double mutant strains. 5KGA production was not observed at all in sldA and double mutant strains. Thus, it seems that "G. dioxyacetonicus" IFO 3271 has another membrane-bound enzyme that reacts with GA, producing 2KGA.