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首页> 外文期刊>Journal of Molecular Biology >The crystal structure of a ternary complex of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa Provides new insight into the reaction mechanism and shows a novel binding mode of the 2'-phosphate of NADP+ and a novel cation binding site.
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The crystal structure of a ternary complex of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa Provides new insight into the reaction mechanism and shows a novel binding mode of the 2'-phosphate of NADP+ and a novel cation binding site.

机译：铜绿假单胞菌甜菜碱醛脱氢酶三元复合物的晶体结构为反应机理提供了新的见识，并显示了NADP + 2'-磷酸的新结合方式和新的阳离子结合位点。

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In the human pathogen Pseudomonas aeruginosa, the NAD(P)(+)-dependent betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine and NADPH, potentially protective against the high-osmolarity and oxidative stresses prevalent in the infected tissues. Disruption of the PaBADH gene negatively affects the growth of bacteria, suggesting that this enzyme could be a target for antibiotic design. PaBADH is one of the few ALDHs that efficiently use NADP(+) and one of the even fewer that require K(+) ions for stability. Crystals of PaBADH were obtained under aerobic conditions in the presence of 2-mercaptoethanol, glycerol, NADP(+) and K(+) ions. The three-dimensional structure was determined at 2.1-A resolution. The catalytic cysteine (C286, corresponding to C302 of ALDH2) is oxidized to sulfenic acid or forms a mixed disulfide with 2-mercaptoethanol. The glutamyl residue involved in the deacylation step (E252, corresponding to E268 of ALDH2) is in two conformations, suggesting a proton relay system formed by two well-conserved residues (E464 and K162, corresponding to E476 and K178, respectively, of ALDH2) that connects E252 with the bulk water. In some active sites, a bound glycerol molecule mimics the thiohemiacetal intermediate; its hydroxyl oxygen is hydrogen bonded to the nitrogen of the amide groups of the side chain of the conserved N153 (N169 of ALDH2) and those of the main chain of C286, which form the oxyanion hole. crystal, and the adenine moiety binds in the usual way. A salt bridge between E179 (E195 of ALDH2) and R40 (E53 of ALDH2) moves the carboxylate group of the former away from the 2'-phosphate of the NADP(+), thus avoiding steric clashes and/or electrostatic repulsion between the two groups. Finally, the crystal shows two K(+) binding sites per subunit. One is in an intrasubunit cavity that we found to be present in all known ALDH structures. The other--not described before for any ALDH but most likely present in most of them--is located in between the dimeric unit, helping structure a region involved in coenzyme binding and catalysis. This may explain the effects of K(+) ions on the activity and stability of PaBADH.
机译：在人类病原体铜绿假单胞菌中，NAD（P）（+）依赖的甜菜碱醛脱氢酶（PaBADH）可能起双重作用，吸收胆碱或胆碱前体的碳和氮（在感染部位富集）并产生甘氨酸甜菜碱。 NADPH和NADPH，可潜在地防止感染组织中普遍存在的高渗透压和氧化应激。 PaBADH基因的破坏对细菌的生长产生负面影响，表明该酶可能是抗生素设计的目标。 PaBADH是有效利用NADP（+）的少数ALDH之一，也是需要K（+）离子以保持稳定性的极少数ALDH之一。 PaBADH晶体是在2-巯基乙醇，甘油，NADP（+）和K（+）离子存在下，在有氧条件下获得的。三维结构是在2.1-A分辨率下确定的。催化半胱氨酸（C286，对应于ALDH2的C302）被氧化为亚磺酸或与2-巯基乙醇形成混合二硫化物。脱酰步骤（E252，对应于ALDH2的E268）涉及的谷氨酰胺残基为两个构象，表明质子传递系统由两个保守性良好的残基（E464和K162，分别对应于ALDH2的E476和K178）形成。将E252与大量水相连。在一些活性位点，结合的甘油分子模拟硫代半缩醛中间体。其羟基氧是氢键合到保守的N153（ALDH2的N169）和C286主链上形成氧阴离子孔的侧链的酰胺基的氮上。晶体，腺嘌呤部分以通常的方式结合。 E179（ALDH2的E195）和R40（ALDH2的E53）之间的盐桥将前者的羧酸酯基移离NADP（+）的2'-磷酸酯，从而避免了两者之间的空间碰撞和/或静电排斥组。最后，晶体显示每个亚基两个K（+）结合位点。一种是在我们发现存在于所有已知的ALDH结构中的亚基内腔中。另一个-以前没有对任何ALDH进行描述，但很可能存在于大多数ALDH中-另一个位于二聚体单元之间，有助于构建参与辅酶结合和催化的区域。这可以解释K（+）离子对PaBADH的活性和稳定性的影响。

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