Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
J Mol Biol. 2010 Sep 3;401(5):906-20. doi: 10.1016/j.jmb.2010.06.069. Epub 2010 Jul 8.
l-Gulonate 3-dehydrogenase (GDH) is a bifunctional dimeric protein that functions not only as an NAD(+)-dependent enzyme in the uronate cycle but also as a taxon-specific lambda-crystallin in rabbit lens. Here we report the first crystal structure of GDH in both apo form and NADH-bound holo form. The GDH protomer consists of two structural domains: the N-terminal domain with a Rossmann fold and the C-terminal domain with a novel helical fold. In the N-terminal domain of the NADH-bound structure, we identified 11 coenzyme-binding residues and found 2 distinct side-chain conformers of Ser124, which is a putative coenzyme/substrate-binding residue. A structural comparison between apo form and holo form and a mutagenesis study with E97Q mutant suggest an induced-fit mechanism upon coenzyme binding; coenzyme binding induces a conformational change in the coenzyme-binding residues Glu97 and Ser124 to switch their activation state from resting to active, which is required for the subsequent substrate recruitment. Subunit dimerization is mediated by numerous intersubunit interactions, including 22 hydrogen bonds and 104 residue pairs of van der Waals interactions, of which those between two cognate C-terminal domains are predominant. From a structure/sequence comparison within GDH homologues, a much greater degree of interprotomer interactions (both polar and hydrophobic) in the rabbit GDH would contribute to its higher thermostability, which may be relevant to the other function of this enzyme as lambda-crystallin, a constitutive structural protein in rabbit lens. The present crystal structures and amino acid mutagenesis studies assigned the role of active-site residues: catalytic base for His145 and substrate binding for Ser124, Cys125, Asn196, and Arg231. Notably, Arg231 participates in substrate binding from the other subunit of the GDH dimer, indicating the functional significance of the dimeric state. Proper orientation of the substrate-binding residues for catalysis is likely to be maintained by an interprotomer hydrogen-bonding network of residues Asn196, Gln199, and Arg231, suggesting a network-based substrate recognition of GDH.
L-古洛糖酸 3-脱氢酶（GDH）是一种具有双功能的二聚体蛋白，不仅在戊糖酸循环中作为 NAD(+)依赖的酶发挥作用，而且在兔晶状体中作为分类特异性的 λ-晶体蛋白发挥作用。在这里，我们报告了 GDH 在 apo 形式和 NADH 结合的全酶形式下的第一个晶体结构。GDH 单体由两个结构域组成：具有 Rossmann 折叠的 N 端结构域和具有新型螺旋折叠的 C 端结构域。在 NADH 结合结构的 N 端结构域中，我们鉴定了 11 个辅酶结合残基，并发现 Ser124 的 2 种独特的侧链构象，Ser124 是一个假定的辅酶/底物结合残基。apo 形式和全酶形式之间的结构比较以及 E97Q 突变体的诱变研究表明，辅酶结合诱导了一种诱导契合机制；辅酶结合诱导辅酶结合残基 Glu97 和 Ser124 的构象变化，将其激活状态从静止变为活跃，这是随后底物募集所必需的。亚基二聚化是通过许多亚基间相互作用介导的，包括 22 个氢键和 104 个残基对范德华相互作用，其中两个同源 C 端结构域之间的相互作用占主导地位。从 GDH 同源物的结构/序列比较来看，兔 GDH 中更多的蛋白间相互作用（既有极性的又有疏水的）有助于其更高的热稳定性，这可能与该酶作为兔晶状体中一种组成型结构蛋白 λ-晶体蛋白的另一种功能有关。目前的晶体结构和氨基酸诱变研究确定了活性位点残基的作用：催化碱为 His145，底物结合残基为 Ser124、Cys125、Asn196 和 Arg231。值得注意的是，Arg231 参与了来自 GDH 二聚体的另一个亚基的底物结合，这表明二聚体状态的功能意义。催化所需的底物结合残基的正确取向可能由 Asn196、Gln199 和 Arg231 残基的蛋白间氢键网络维持，这表明 GDH 的基于网络的底物识别。
