Li Zack Z, Riegert Alexander S, Goneau Marie-France, Cunningham Anna M, Vinogradov Evgeny, Li Jianjun, Schoenhofen Ian C, Thoden James B, Holden Hazel M, Gilbert Michel
National Research Council Canada, Human Health Therapeutics, 100 Sussex Drive, Ottawa, ON, Canada.
Department of Biochemistry, University of Wisconsin, 440 Henry Mall, Madison, WI, USA.
Glycobiology. 2017 Apr 1;27(4):358-369. doi: 10.1093/glycob/cww136.
The Gram-negative bacterium Campylobacter jejuni 81116 (Penner serotype HS:6) has a class E lipooligosaccharide (LOS) biosynthesis locus containing 19 genes, which encode for 11 putative glycosyltransferases, 1 lipid A acyltransferase and 7 enzymes thought to be involved in the biosynthesis of dideoxyhexosamine (ddHexN) moieties. Although the LOS outer core structure of C. jejuni 81116 is still unknown, recent mass spectrometry analyses suggest that it contains acetylated forms of two ddHexN residues. For this investigation, five of the genes encoding enzymes reportedly involved in the biosyntheses of these sugar residues were examined, rmlA, rmlB, wlaRA, wlaRB and wlaRG. Specifically, these genes were cloned and expressed in Escherichia coli, and the corresponding enzymes were purified and tested for biochemical activity. Here we present data demonstrating that RmlA functions as a glucose-1-phosphate thymidylyltransferase and that RmlB is a thymidine diphosphate (dTDP)-glucose 4,6-dehydratase. We also show, through nuclear magnetic resonance spectroscopy and mass spectrometry analyses, that WlaRG, when utilized in coupled assays with either WlaRA or WlaRB and dTDP-4-keto-6-deoxyglucose, results in the production of either dTDP-3-amino-3,6-dideoxy-d-galactose (dTDP-Fuc3N) or dTDP-3-amino-3,6-dideoxy-d-glucose (dTDP-Qui3N), respectively. In addition, the X-ray crystallographic structures of the 3,4-ketoisomerases, WlaRA and WlaRB, were determined to 2.14 and 2.0 Å resolutions, respectively. Taken together, the data reported herein demonstrate that C. jejuni 81116 utilizes five enzymes to synthesize dTDP-Fuc3N or dTDP-Qui3N and that WlaRG, an aminotransferase, can function on sugars with differing stereochemistry about their C-4' carbons. Importantly, the data reveal that C. jejuni 81116 has the ability to synthesize two isomeric ddHexN forms.
革兰氏阴性菌空肠弯曲菌81116（彭纳血清型HS:6）具有一个E类脂寡糖（LOS）生物合成基因座，包含19个基因，这些基因编码11种假定的糖基转移酶、1种脂多糖A酰基转移酶以及7种被认为参与二脱氧己糖胺（ddHexN）部分生物合成的酶。尽管空肠弯曲菌81116的LOS外核结构仍不清楚，但最近的质谱分析表明，它含有两种ddHexN残基的乙酰化形式。在本研究中，检测了据报道参与这些糖残基生物合成的5个酶编码基因，即rmlA、rmlB、wlaRA、wlaRB和wlaRG。具体而言，将这些基因克隆并在大肠杆菌中表达，纯化相应的酶并测试其生化活性。在此，我们展示的数据表明，RmlA作为葡萄糖-1-磷酸胸苷酰转移酶发挥作用，而RmlB是胸苷二磷酸（dTDP）-葡萄糖4,6-脱水酶。我们还通过核磁共振光谱和质谱分析表明，当WlaRG与WlaRA或WlaRB以及dTDP-4-酮-6-脱氧葡萄糖进行偶联测定时，分别产生dTDP-3-氨基-3,6-二脱氧-D-半乳糖（dTDP-Fuc3N）或dTDP-3-氨基-3,6-二脱氧-D-葡萄糖（dTDP-Qui3N）。此外，3,4-酮异构酶WlaRA和WlaRB的X射线晶体结构分别确定为2.14 Å和2.0 Å分辨率。综上所述，本文报道的数据表明，空肠弯曲菌81116利用五种酶合成dTDP-Fuc3N或dTDP-Qui3N，并且氨基转移酶WlaRG可以作用于其C-4'碳具有不同立体化学的糖。重要的是，数据显示空肠弯曲菌81116有能力合成两种异构的ddHexN形式。
