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首页> 外文期刊>Biochemistry >X-ray Structure of KijD3, a Key Enzyme Involved in the Biosynthesis of d-Kijanose
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X-ray Structure of KijD3, a Key Enzyme Involved in the Biosynthesis of d-Kijanose

机译：KijD3的X射线结构，一种参与D-Kijanose生物合成的关键酶

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摘要

D-Kijanose is an unusual nitrosugar found attached to the antibiotic kijanimicin. Ten enzymes arenrequired for its production in Actinomadura kijaniata, a soil-dwelling actinomycete. The focus of thisninvestigation is on the protein encoded by the kijd3 gene and hereafter referred to as KijD3. On the basis ofnamino acid sequence analyses, KijD3 has been proposed to be an FAD-dependent oxidoreductase, whichncatalyzes the sixth step in D-kijanose biosynthesis by converting dTDP-3-amino-2,3,6-trideoxy-4-keto-3-nmethyl-D-glucose into its C-30nnitro derivative. This putative activity, however, has never been demonstratednin vivo or in vitro. Here we report the first structural study of this enzyme. For our investigation, crystals ofKijD3nwere grown in the presence of dTDP, and the structure was solved to 2.05-A resolution. The enzyme is a tetramernwith each subunit folding into three distinct regions: a five R-helical bundle, an eight-stranded β-sheet, and ansecond five R-helical bundle. The dTDP moiety is anchored to the protein via the side chains of Glu 113, Gln 254,nand Arg 330. The overall fold of KijD3 places it into the well-characterized fatty acyl-CoA dehydrogenasensuperfamily. There is a decided cleft in each subunit with the appropriate dimensions to accommodate a dTDP-nlinked sugar. Strikingly, the loop defined by Phe 383 to Ala 388, which projects into the active site, contains twonadjacent cis-peptide bonds, Pro 386 and Tyr 387.Activity assays demonstrate thatKijD3 requires FADfor activitynand that it produces a hydroxylamino product. Themolecular architecture ofKijD3 described in this report servesnas a paradigm for a new family of enzymes that function on dTDP-linked sugar substrates.
机译：D-Kijanose是发现与抗生素kijanimicin结合的不寻常的硝基糖。在居住在土壤中的放线菌Actinomadura kijaniata中需要十种酶来生产。这项研究的重点是由kijd3基因编码的蛋白质，以下称为KijD3。根据氨基酸序列分析，有人提出KijD3是一种FAD依赖的氧化还原酶，它通过转化dTDP-3-amino-2,3,6-trideoxy-4-keto-3来催化D-木糖生物合成的第六步。 -n甲基-D-葡萄糖转化为其C-30nnitro衍生物。然而，从未在体内或体外证明过这种推定的活性。在这里，我们报告这种酶的首次结构研究。为了进行研究，在dTDP存在下生长了KijD3n晶体，并将结构解析为2.05-A分辨率。该酶是四聚体，每个亚基折叠成三个不同的区域：五个R螺旋束，一个八链β-折叠和另一个五个R螺旋束。 dTDP部分通过Glu 113，Gln 254，n和Arg 330的侧链锚定到蛋白质上。KijD3的整体折叠将其置于特征明确的脂肪酰基CoA脱氢酶超家族中。每个亚基中都有一个确定的裂口，具有适当的尺寸，可以容纳dTDP-n连接的糖。令人惊讶的是，由Phe 383定义到Ala 388的环（伸入活性位点）包含两个相邻的顺式肽键Pro 386和Tyr387。活性测定表明，KijD3需要FAD才能产生活性，并且它会产生羟氨基​​产物。本报告中描述的KijD3的分子结构为在dTDP连接的糖底物上起作用的新的酶家族提供了范例。

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