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首页> 外文期刊>Journal of Molecular Biology >Structural and Biochemical Characterization of a Novel Aldehyde Dehydrogenase Encoded by the Benzoate Oxidation Pathway in Burkholderia xenovorans LB400
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Structural and Biochemical Characterization of a Novel Aldehyde Dehydrogenase Encoded by the Benzoate Oxidation Pathway in Burkholderia xenovorans LB400

机译：伯克霍尔德菌xenovorans LB400中苯甲酸酯氧化途径编码的新型醛脱氢酶的结构和生化特性

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The recently identified benzoate oxidation (box) pathway in Burkholderia xenovorans LB400 (LB400 hereinafter) assimilates benzoate through a unique mechanism where each intermediate is processed as a coenzyme A (CoA) thioester. A key step in this process is the conversion of 3,4-dehydroadipyl-CoA semialdehyde into its corresponding CoA acid by a novel aldehyde dehydrogenase (ALDH) (EC 1.2.1.x). The goal of this study is to characterize the biochemical and structural properties of the chromosomally encoded form of this new class of ALDHs from LB400 (ALDHC) in order to better understand its role in benzoate degradation. To this end, we carried out kinetic studies with six structurally diverse aldehydes and nicotinamide adenine dinucleotide (phosphate) (NAD + and NADP +). Our data definitively show that ALDHC is more active in the presence of NADP + and selective for linear medium-chain to long-chain aldehydes. To elucidate the structural basis for these biochemical observations, we solved the 1.6-? crystal structure of ALDHC in complex with NADPH bound in the cofactor-binding pocket and an ordered fragment of a polyethylene glycol molecule bound in the substrate tunnel. These data show that cofactor selectivity is governed by a complex network of hydrogen bonds between the oxygen atoms of the 2′-phosphoryl moiety of NADP + and a threonine/lysine pair on ALDHC. The catalytic preference of ALDHC for linear longer-chain substrates is mediated by a deep narrow configuration of the substrate tunnel. Comparative analysis reveals that reorientation of an extended loop (Asn478-Pro490) in ALDHC induces the constricted structure of the substrate tunnel, with the side chain of Asn478 imposing steric restrictions on branched-chain and aromatic aldehydes. Furthermore, a key glycine (Gly104) positioned at the mouth of the tunnel allows for maximum tunnel depth required to bind medium-chain to long-chain aldehydes. This study provides the first integrated biochemical and structural characterization of a box-pathway-encoded ALDH from any organism and offers insight into the catalytic role of ALDHC in benzoate degradation.
机译：最近鉴定的异种伯克霍尔德氏菌LB400（下文称为LB400）中的苯甲酸酯氧化（盒）途径通过独特的机理同化苯甲酸酯，其中每种中间体均作为辅酶A（CoA）硫酯加工。此过程中的关键步骤是通过新型醛脱氢酶（ALDH）（EC 1.2.1.x）将3,4-脱氢己二酰-CoA半醛转化为其相应的CoA酸。这项研究的目的是表征来自LB400（ALDHC）的新型ALDH的染色体编码形式的生化和结构特性，以更好地了解其在苯甲酸酯降解中的作用。为此，我们对六个结构不同的醛和烟酰胺腺嘌呤二核苷酸（磷酸）（NAD +和NADP +）进行了动力学研究。我们的数据明确表明，在NADP +存在下，ALDHC更具活性，对线性中链至长链醛具有选择性。为了阐明这些生化观察的结构基础，我们解决了1.6-？ ALDHC的晶体结构与结合在辅因子结合袋中的NADPH和结合在底物通道中的聚乙二醇分子的有序片段复合。这些数据表明，辅因子的选择性由NADP +的2'-磷酰基部分的氧原子与ALDHC上的苏氨酸/赖氨酸对之间的氢键的复杂网络决定。线性长链底物对ALDHC的催化偏好是由底物通道的深窄构型介导的。对比分析表明，ALDHC中延长环（Asn478-Pro490）的重新取向会诱导底物通道的收缩结构，而Asn478的侧链对支链和芳族醛施加空间限制。此外，位于通道口的关键甘氨酸（Gly104）允许将中链醛与长链醛键合所需的最大通道深度。这项研究提供了来自任何生物的盒途径编码的ALDH的第一个综合的生化和结构表征，并提供了对ALDHC在苯甲酸酯降解中的催化作用的见解。

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