Toyama Hirohide, Chen Zhi-Wei, Fukumoto Megumi, Adachi Osao, Matsushita Kazunobu, Mathews F Scott
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan.
J Mol Biol. 2005 Sep 9;352(1):91-104. doi: 10.1016/j.jmb.2005.06.078.
Depending on the alcohols used as growth substrates, Pseudomonas putida HK5 produces two distinct quinohemoprotein alcohol dehydrogenases, ADH-IIB and ADH-IIG, both of which contain pyrroloquinoline quinone (PQQ) and heme c as the prosthetic groups but show different substrate specificities, especially for diol substrates. Molecular cloning of the gene of ADH-IIB and its crystal structure are already reported. Here, molecular cloning of the gene, qgdA, and solution of the three-dimensional structure of ADH-IIG are reported. The enzyme consists of 718 amino acid residues including a signal sequence of 29 amino acid residues. The PQQ domain is highly homologous to other quinoproteins, especially to quinohemoproteins. The crystal structure of ADH-IIG, determined at 2.2A resolution, shows that the overall structure and the amino acid residues involved in PQQ binding are quite similar to ADH-IIB and to another quinohemoprotein ADH, qhEDH from Comamonas testosteroni. However, the lengths of the linker regions connecting the PQQ and the cytochrome domains are different from each other, leading to a significant difference in orientation of the cytochrome domain with respect to the PQQ domain. Apart from ADH-IIB and qhEDH, ADH-IIG has an extra 12-residue helix within loop 3 in the PQQ domain and an extra 3(10) helix in the C terminus of the cytochrome domain, and both helices appear parallel and linked by a hydrogen bond. The amino acid residues contacting substrate/product in the crystal structures are also different among them. In the crystal structure of ADH-IIG with 1,2-propanediol, one of the hydroxyl groups of the substrate forms a hydrogen bond with O5 of PQQ and OD1 of Asp300, and the other interacts with a water molecule and with NE2 of Trp386, the corresponding residue of which is not found in ADH-IIB and qhEDH, and might be the residue responsible for making ADH-IIG prefer diol substrates.
根据用作生长底物的醇类不同，恶臭假单胞菌HK5可产生两种不同的醌血红蛋白醇脱氢酶，即ADH-IIB和ADH-IIG，二者均含有吡咯喹啉醌（PQQ）和血红素c作为辅基，但表现出不同的底物特异性，尤其是对二醇底物。ADH-IIB基因的分子克隆及其晶体结构已有报道。在此，报道了ADH-IIG基因qgdA的分子克隆及其三维结构解析。该酶由718个氨基酸残基组成，包括一个29个氨基酸残基的信号序列。PQQ结构域与其他醌蛋白高度同源，尤其是与醌血红蛋白。以2.2 Å分辨率测定的ADH-IIG晶体结构表明，其整体结构以及参与PQQ结合的氨基酸残基与ADH-IIB和另一种醌血红蛋白ADH（睾丸酮丛毛单胞菌的qhEDH）非常相似。然而，连接PQQ和细胞色素结构域的连接区长度彼此不同，导致细胞色素结构域相对于PQQ结构域的方向存在显著差异。除了ADH-IIB和qhEDH，ADH-IIG在PQQ结构域的环3内有一个额外的12个残基的螺旋，在细胞色素结构域的C末端有一个额外的3(10)螺旋，且两个螺旋呈平行排列并通过氢键相连。它们在晶体结构中与底物/产物接触的氨基酸残基也不同。在与1,2-丙二醇结合的ADH-IIG晶体结构中，底物的一个羟基与PQQ的O5以及Asp300的OD1形成氢键，另一个羟基与一个水分子以及Trp386的NE2相互作用，ADH-IIB和qhEDH中未发现其相应残基，该残基可能是使ADH-IIG偏好二醇底物的原因。
