Brockhausen Inka, Nair Dileep G, Chen Min, Yang Xiaojing, Allingham John S, Szarek Walter A, Anastassiades Tassos
a Department of Medicine, Division of Rheumatology, Queen's University, Kingston, ON K7L 3N6, Canada.
b Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada.
Biochem Cell Biol. 2016 Apr;94(2):197-204. doi: 10.1139/bcb-2015-0115. Epub 2015 Dec 15.
Glucosamine-6-phosphate N-acetyltransferase1 (GNA1) catalyses the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to glucosamine-6-phosphate (GlcN6P) to form N-acetylglucosamine-6-phosphate (GlcNAc6P), which is an essential intermediate in UDP-GlcNAc biosynthesis. An analog of GlcNAc, N-butyrylglucosamine (GlcNBu) has shown healing properties for bone and articular cartilage in animal models of arthritis. The goal of this work was to examine whether GNA1 has the ability to transfer a butyryl group from butyryl-CoA to GlcN6P to form GlcNBu6P, which can then be converted to GlcNBu. We developed fluorescent and radioactive assays and examined the donor specificity of human GNA1. Acetyl, propionyl, n-butyryl, and isobutyryl groups were all transferred to GlcN6P, but isovaleryl-CoA and decanoyl-CoA did not serve as donor substrates. Site-specific mutants were produced to examine the role of amino acids potentially affecting the size and properties of the AcCoA binding pocket. All of the wild type and mutant enzymes showed activities of both acetyl and butyryl transfer and can therefore be used for the enzymatic synthesis of GlcNBu for biomedical applications.
6-磷酸葡糖胺N-乙酰基转移酶1（GNA1）催化将乙酰基从乙酰辅酶A（AcCoA）转移至6-磷酸葡糖胺（GlcN6P），形成N-乙酰葡糖胺-6-磷酸（GlcNAc6P），后者是UDP-GlcNAc生物合成中的关键中间体。N-丁酰葡糖胺（GlcNBu）作为GlcNAc的类似物，已在关节炎动物模型中显示出对骨骼和关节软骨的修复特性。这项工作的目标是研究GNA1是否有能力将丁酰基从丁酰辅酶A转移至GlcN6P，形成GlcNBu6P，然后再将其转化为GlcNBu。我们开发了荧光和放射性检测方法，并研究了人GNA1的供体特异性。乙酰基、丙酰基、正丁酰基和异丁酰基均能转移至GlcN6P，但异戊酰辅酶A和癸酰辅酶A不作为供体底物。构建了位点特异性突变体，以研究可能影响AcCoA结合口袋大小和性质的氨基酸的作用。所有野生型和突变型酶均显示出乙酰基和丁酰基转移活性，因此可用于生物医学应用中GlcNBu的酶促合成。
