Department of Biochemistry, University of Wisconsin, Madison, Wisconsin, USA.
Protein Sci. 2021 Sep;30(9):1882-1894. doi: 10.1002/pro.4139. Epub 2021 Jun 21.
Acanthamoeba polyphaga Mimivirus, a complex virus that infects amoeba, was first reported in 2003. It is now known that its DNA genome encodes for nearly 1,000 proteins including enzymes that are required for the biosynthesis of the unusual sugar 4-amino-4,6-dideoxy-d-glucose, also known as d-viosamine. As observed in some bacteria, the pathway for the production of this sugar initiates with a nucleotide-linked sugar, which in the Mimivirus is thought to be UDP-d-glucose. The enzyme required for the installment of the amino group at the C-4' position of the pyranosyl moiety is encoded in the Mimivirus by the L136 gene. Here, we describe a structural and functional analysis of this pyridoxal 5'-phosphate-dependent enzyme, referred to as L136. For this analysis, three high-resolution X-ray structures were determined: the wildtype enzyme/pyridoxamine 5'-phosphate/dTDP complex and the site-directed mutant variant K185A in the presence of either UDP-4-amino-4,6-dideoxy-d-glucose or dTDP-4-amino-4,6-dideoxy-d-glucose. Additionally, the kinetic parameters of the enzyme utilizing either UDP-d-glucose or dTDP-d-glucose were measured and demonstrated that L136 is efficient with both substrates. This is in sharp contrast to the structurally related DesI from Streptomyces venezuelae, whose three-dimensional architecture was previously reported by this laboratory. As determined in this investigation, DesI shows a profound preference in its catalytic efficiency for the dTDP-linked sugar substrate. This difference can be explained in part by a hydrophobic patch in DesI that is missing in L136. Notably, the structure of L136 reported here represents the first three-dimensional model for a virally encoded PLP-dependent enzyme and thus provides new information on sugar aminotransferases in general.
多噬棘阿米巴曼氏病毒（Acanthamoeba polyphaga Mimivirus），一种感染变形虫的复杂病毒，于 2003 年首次报道。现在已知，其 DNA 基因组编码近 1000 种蛋白质，包括合成异常糖 4-氨基-4,6-二脱氧-d-葡萄糖（也称为 d-卫矛醇）所需的酶。与某些细菌一样，该糖的生物合成途径始于核苷酸连接的糖，在 Mimivirus 中，该糖被认为是 UDP-d-葡萄糖。在吡喃糖部分的 C-4'位置安装氨基基团所需的酶由 Mimivirus 中的 L136 基因编码。在这里，我们描述了这种依赖于吡哆醛 5'-磷酸的酶，称为 L136 的结构和功能分析。为此分析，确定了三个高分辨率 X 射线结构：野生型酶/吡哆胺 5'-磷酸/dTDP 复合物和存在 UDP-4-氨基-4,6-二脱氧-d-葡萄糖或 dTDP-4-氨基-4,6-二脱氧-d-葡萄糖时的定点突变变体 K185A。此外，还测量了该酶利用 UDP-d-葡萄糖或 dTDP-d-葡萄糖的动力学参数，并证明 L136 对两种底物都有效。这与结构上相关的委内瑞拉链霉菌的 DesI 形成鲜明对比，该实验室以前曾报道过其三维结构。如本研究中所确定的，DesI 对其催化效率表现出对 dTDP 连接的糖底物的明显偏好。这种差异部分可以通过 DesI 中缺少疏水区来解释，而 L136 中则缺少疏水区。值得注意的是，这里报道的 L136 结构代表了第一个病毒编码的 PLP 依赖性酶的三维模型，因此为一般的糖氨基转移酶提供了新的信息。
