背景:氧化葡糖酸杆菌,被用于生物技术,因为它能够氧化各种各样的碳水化合物,酒精,和多元醇通过位于周质空间中的膜结合脱氢酶以立体和区域选择性的方式。这些反应遵循众所周知的伯特兰-哈德森规则。在我们之前的研究中(BBA-一般受试者,2021年,1865年:129740),我们发现葡糖杆菌属,包括氧化双歧杆菌和cerinus菌株可以区域选择性地将D-半乳糖醇的C-3和C-5羟基氧化为稀有糖D-塔格糖和L-木-3-己糖,这代表了BertrandHudson规则的例外。催化该反应的酶位于周质空间或膜结合,并且是PQQ(吡咯并喹啉奎宁)和Ca2依赖性的;鼓励我们确定哪种类型的酶催化这种独特的反应。
方法:通过将氧化葡糖杆菌621H的多缺失菌株与所有推定的膜结合脱氢酶基因互补来鉴定酶。
结论:在这项研究中,我们确定了该基因编码膜结合的PQQ依赖性脱氢酶,该脱氢酶在其3'-OH和5'-OH中催化独特的半乳糖醇氧化反应。多缺失氧化葡糖BP.9菌株中的补体实验确定,酶mSLDH(由GOX0855-0854编码,sldB-sldA)负责半乳糖醇的独特氧化反应。此外,我们证明了mSLDH的小亚基SldB是膜结合的,并通过将其与红色荧光蛋白(mRubby)融合而充当锚定蛋白,并在大肠杆菌和酵母解脂耶氏酵母中异源表达。需要SldB亚基来维持催化D-半乳糖醇转化为L-木-3-己酮糖和D-塔格糖的全酶活性。由GOX0854编码的大亚基SldA也被表征,发现其24个氨基酸的信号肽是mSLDH蛋白脱氢活性所必需的。
结论:在这项研究中,主要的膜结合多元醇脱氢酶mSLDH在氧化黑曲霉621H被证明是催化独特的半乳糖醇氧化,这代表了BertrandHudson规则的例外,并拓宽了mSLDH的底物范围。进一步破译明确的酶机理将证明这一理论。
Gluconobacter oxydans, is used in biotechnology because of its ability to oxidize a wide variety of carbohydrates, alcohols, and polyols in a stereo- and regio-selective manner by membrane-bound dehydrogenases located in periplasmic space. These reactions obey the well-known Bertrand-Hudson\'s rule. In our previous study (BBA-General Subjects, 2021, 1865:129740), we discovered that Gluconobacter species, including G. oxydans and G. cerinus strain can regio-selectively oxidize the C-3 and C-5 hydroxyl groups of D-galactitol to rare sugars D-tagatose and L-xylo-3-hexulose, which represents an exception to Bertrand Hudson\'s rule. The enzyme catalyzing this reaction is located in periplasmic space or membrane-bound and is PQQ (pyrroloquinoline quinine) and Ca2+-dependent; we were encouraged to determine which type of enzyme(s) catalyze this unique reaction.
Enzyme was identified by complementation of multi-deletion strain of Gluconobacter oxydans 621H with all putative membrane-bound dehydrogenase genes.
In this study, we identified this gene encoding the membrane-bound PQQ-dependent dehydrogenase that catalyzes the unique galactitol oxidation reaction in its 3\'-OH and 5\'-OH. Complement experiments in multi-deletion G. oxydans BP.9 strains established that the enzyme mSLDH (encoded by GOX0855-0854, sldB-sldA) is responsible for galactitol\'s unique oxidation reaction. Additionally, we demonstrated that the small subunit SldB of mSLDH was membrane-bound and served as an anchor protein by fusing it to a red fluorescent protein (mRubby), and heterologously expressed in E. coli and the yeast Yarrowia lipolytica. The SldB subunit was required to maintain the holo-enzymatic activity that catalyzes the conversion of D-galactitol to L-xylo-3-hexulose and D-tagatose. The large subunit SldA encoded by GOX0854 was also characterized, and it was discovered that its 24 amino acids signal peptide is required for the dehydrogenation activity of the mSLDH protein.
In this study, the main membrane-bound polyol dehydrogenase mSLDH in G. oxydans 621H was proved to catalyze the unique galactitol oxidation, which represents an exception to the Bertrand Hudson\'s rule, and broadens its substrate ranges of mSLDH. Further deciphering the explicit enzymatic mechanism will prove this theory.