Olivier Nelson B, Chen Mark M, Behr Jonathan R, Imperiali Barbara
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Biochemistry. 2006 Nov 14;45(45):13659-69. doi: 10.1021/bi061456h.
In Campylobacter jejuni 2,4-diacetamido-2,4,6-trideoxy-alpha-d-glucopyranose, termed N,N'-diacetylbacillosamine (Bac2,4diNAc), is the first carbohydrate in the glycoprotein N-linked heptasaccharide. With uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) as a starting point, two enzymes of the general protein glycosylation (Pgl) pathway in C. jejuni (PglF and PglE) have recently been shown to modify this sugar nucleotide to form UDP-2-acetamido-4-amino-2,4,6-trideoxy-alpha-d-glycopyranose (UDP-4-amino-sugar) [Schoenhofen, I. C., et al. (2006) J. Biol. Chem. 281, 723-732]. PglD has been proposed to catalyze the final step in N,N'-diacetylbacillosamine synthesis by N-acetylation of the UDP-4-amino-sugar at the C4 position. We have cloned, overexpressed, and purified PglD from the pgl locus of C. jejuni NCTC 11168 and identified it as the acetyltransferase that modifies the UDP-4-amino-sugar to form UDP-N,N'-diacetylbacillosamine, utilizing acetyl-coenzyme A as the acetyl group donor. The UDP-N,N'-diacetylbacillosamine product was purified from the reaction by reverse phase C18 HPLC and the structure determined by NMR analysis. Additionally, the full-length PglF was overexpressed and purified in the presence of detergent as a GST fusion protein, allowing for derivation of kinetic parameters. We found that the UDP-4-amino-sugar was readily synthesized from UDP-GlcNAc in a coupled reaction using PglF and PglE. We also demonstrate the in vitro biosynthesis of the complete heptasaccharide lipid-linked donor by coupling the action of eight enzymes (PglF, PglE, PglD, PglC, PglA, PglJ, PglH, and PglI) in the Pgl pathway in a single reaction vessel.
在空肠弯曲杆菌中，2,4 - 二乙酰氨基 - 2,4,6 - 三脱氧 -α - D - 吡喃葡萄糖，即N,N'-二乙酰杆菌糖胺（Bac2,4diNAc），是糖蛋白N - 连接七糖中的首个碳水化合物。以尿苷二磷酸 - N - 乙酰葡糖胺（UDP - GlcNAc）为起始点，空肠弯曲杆菌中一般蛋白质糖基化（Pgl）途径的两种酶（PglF和PglE）最近已被证明可修饰这种糖核苷酸，形成UDP - 2 - 乙酰氨基 - 4 - 氨基 - 2,4,6 - 三脱氧 -α - D - 吡喃葡萄糖（UDP - 4 - 氨基糖）[舍恩霍芬，I. C.等人（2006年）《生物化学杂志》281卷，723 - 732页]。有人提出PglD通过在C4位置对UDP - 4 - 氨基糖进行N - 乙酰化来催化N,N'-二乙酰杆菌糖胺合成的最后一步。我们从空肠弯曲杆菌NCTC 11168的pgl基因座克隆、过量表达并纯化了PglD，并确定它是以乙酰辅酶A作为乙酰基供体，将UDP - 4 - 氨基糖修饰形成UDP - N,N'-二乙酰杆菌糖胺的乙酰转移酶。通过反相C18高效液相色谱从反应中纯化出UDP - N,N'-二乙酰杆菌糖胺产物，并通过核磁共振分析确定其结构。此外，全长PglF在去污剂存在下作为GST融合蛋白进行了过量表达和纯化，从而可以推导动力学参数。我们发现UDP - 4 - 氨基糖在使用PglF和PglE的偶联反应中很容易从UDP - GlcNAc合成。我们还通过在单个反应容器中偶联Pgl途径中的八种酶（PglF、PglE、PglD、PglC、PglA、PglJ、PglH和PglI）的作用，证明了完整七糖脂质连接供体的体外生物合成。
