Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Biochemistry. 2010 May 4;49(17):3517-24. doi: 10.1021/bi100318v.
D-kijanose is an unusual nitrosugar found attached to the antibiotic kijanimicin. Ten enzymes are required for its production in Actinomadura kijaniata, a soil-dwelling actinomycete. The focus of this investigation is on the protein encoded by the kijd3 gene and hereafter referred to as KijD3. On the basis of amino acid sequence analyses, KijD3 has been proposed to be an FAD-dependent oxidoreductase, which catalyzes the sixth step in d-kijanose biosynthesis by converting dTDP-3-amino-2,3,6-trideoxy-4-keto-3-methyl-d-glucose into its C-3' nitro derivative. This putative activity, however, has never been demonstrated in vivo or in vitro. Here we report the first structural study of this enzyme. For our investigation, crystals of KijD3 were grown in the presence of dTDP, and the structure was solved to 2.05-A resolution. The enzyme is a tetramer with each subunit folding into three distinct regions: a five alpha-helical bundle, an eight-stranded beta-sheet, and a second five alpha-helical bundle. The dTDP moiety is anchored to the protein via the side chains of Glu 113, Gln 254, and Arg 330. The overall fold of KijD3 places it into the well-characterized fatty acyl-CoA dehydrogenase superfamily. There is a decided cleft in each subunit with the appropriate dimensions to accommodate a dTDP-linked sugar. Strikingly, the loop defined by Phe 383 to Ala 388, which projects into the active site, contains two adjacent cis-peptide bonds, Pro 386 and Tyr 387. Activity assays demonstrate that KijD3 requires FAD for activity and that it produces a hydroxylamino product. The molecular architecture of KijD3 described in this report serves as a paradigm for a new family of enzymes that function on dTDP-linked sugar substrates.
D-赤藓糖是一种不寻常的亚硝糖，与抗生素奇霉素结合。在土壤放线菌 Actinomadura kijaniata 中，需要十种酶才能产生 D-赤藓糖。本研究的重点是由 kij3 基因编码的蛋白质，此后称为 KijD3。根据氨基酸序列分析，KijD3 被提议为 FAD 依赖性氧化还原酶，通过将 dTDP-3-氨基-2,3,6-三脱氧-4-酮-3-甲基-d-葡萄糖转化为其 C-3'硝基衍生物，催化 D-赤藓糖生物合成的第六步。然而，这种假定的活性从未在体内或体外得到证明。在这里，我们报告了该酶的首次结构研究。为了进行我们的研究，在存在 dTDP 的情况下培养了 KijD3 的晶体，并将结构解析至 2.05-A 的分辨率。该酶是一个四聚体，每个亚基折叠成三个不同的区域：一个五螺旋束、一个八链β-折叠和第二个五螺旋束。dTDP 部分通过 Glu 113、Gln 254 和 Arg 330 的侧链与蛋白质结合。KijD3 的整体折叠将其归入特征明确的脂肪酸辅酶 A 脱氢酶超家族。每个亚基中都有一个明显的裂缝，其尺寸适合容纳与 dTDP 相连的糖。引人注目地，由 Phe 383 到 Ala 388 定义的环，突入活性部位，包含两个相邻的顺式肽键，Pro 386 和 Tyr 387。活性测定表明 KijD3 需要 FAD 才能发挥活性，并产生羟基氨基产物。本报告中描述的 KijD3 的分子结构为作用于 dTDP 连接糖底物的新酶家族提供了范例。
