Deacon A M, Ni Y S, Coleman W G, Ealick S E
Department ofChemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
Structure. 2000 May 15;8(5):453-62. doi: 10.1016/s0969-2126(00)00128-3.
ADP-L-glycero--mannoheptose 6-epimerase (AGME) is required for lipopolysaccharide (LPS) biosynthesis in most genera of pathogenic and non-pathogenic Gram-negative bacteria. It catalyzes the interconversion of ADP-D-glycero-D-mannoheptose and ADP-L-glycero-D-mannoheptose, a precursor of the seven-carbon sugar L-glycero-mannoheptose (heptose). Heptose is an obligatory component of the LPS core domain; its absence results in a truncated LPS structure resulting in susceptibility to hydrophobic antibiotics. Heptose is not found in mammalian cells, thus its biosynthetic pathway in bacteria presents a unique target for the design of novel antimicrobial agents.
The structure of AGME, in complex with NADP and the catalytic inhibitor ADP-glucose, has been determined at 2.0 A resolution by multiwavelength anomalous diffraction (MAD) phasing methods. AGME is a homopentameric enzyme, which crystallizes with two pentamers in the asymmetric unit. The location of 70 crystallographically independent selenium sites was a key step in the structure determination process. Each monomer comprises two domains: a large N-terminal domain, consisting of a modified seven-stranded Rossmann fold that is associated with NADP binding; and a smaller alpha/beta C-terminal domain involved in substrate binding.
The first structure of an LPS core biosynthetic enzyme leads to an understanding of the mechanism of the conversion between ADP-D-glycero--mannoheptose and ADP-L-glycero-D-mannoheptose. On the basis of its high structural similarity to UDP-galactose epimerase and the three-dimensional positions of the conserved residues Ser116, Tyr140 and Lys144, AGME was classified as a member of the short-chain dehydrogenase/reductase (SDR) superfamily. This study should prove useful in the design of mechanistic and structure-based inhibitors of the AGME catalyzed reaction.
ADP-L-甘油-D-甘露庚糖6-表异构酶（AGME）是大多数致病性和非致病性革兰氏阴性菌中脂多糖（LPS）生物合成所必需的。它催化ADP-D-甘油-D-甘露庚糖和ADP-L-甘油-D-甘露庚糖（七碳糖L-甘油-D-甘露庚糖（庚糖）的前体）之间的相互转化。庚糖是LPS核心结构域的必需成分；其缺失会导致LPS结构截短，从而使细菌对疏水性抗生素敏感。哺乳动物细胞中不存在庚糖，因此其在细菌中的生物合成途径是设计新型抗菌剂的独特靶点。
通过多波长反常衍射（MAD）相位法，以2.0埃分辨率测定了与NADP和催化抑制剂ADP-葡萄糖结合的AGME的结构。AGME是一种同五聚体酶，在不对称单元中与两个五聚体一起结晶。70个晶体学独立硒位点的定位是结构测定过程中的关键步骤。每个单体包含两个结构域：一个大的N端结构域，由与NADP结合相关的修饰七链Rossmann折叠组成；以及一个较小的参与底物结合的α/β C端结构域。
LPS核心生物合成酶的首个结构有助于理解ADP-D-甘油-D-甘露庚糖和ADP-L-甘油-D-甘露庚糖之间的转化机制。基于其与UDP-半乳糖表异构酶的高度结构相似性以及保守残基Ser116、Tyr140和Lys（赖氨酸）144的三维位置，AGME被归类为短链脱氢酶/还原酶（SDR）超家族的成员。该研究应有助于设计基于机制和结构的AGME催化反应抑制剂。
