Zahniser Megan P D, Prasad Shreenath, Kneen Malea M, Kreinbring Cheryl A, Petsko Gregory A, Ringe Dagmar, McLeish Michael J
Department of Biochemistry, Brandeis University, 415 South St., Waltham, MA 02454,USA.
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis (IUPUI), 402 N. Blackford Street, Indianapolis, IN 46202,USA.
Protein Eng Des Sel. 2017 Mar 1;30(3):271-278. doi: 10.1093/protein/gzx015.
Benzaldehyde dehydrogenase from Pseudomonas putida (PpBADH) belongs to the Class 3 aldehyde dehydrogenase (ALDH) family. The Class 3 ALDHs are unusual in that they are generally dimeric (rather than tetrameric), relatively non-specific and utilize both NAD+ and NADP+. To date, X-ray structures of three Class 3 ALDHs have been determined, of which only two have cofactor bound, both in the NAD+ form. Here we report the crystal structure of PpBADH in complex with NADP+ and a thioacyl intermediate adduct. The overall architecture of PpBADH resembles that of most other members of the ALDH superfamily, and the cofactor binding residues are well conserved. Conversely, the pattern of cofactor binding for the rat Class 3 ALDH differs from that of PpBADH and other ALDHs. This has been interpreted in terms of a different mechanism for the rat enzyme. Comparison with the PpBADH structure, as well as multiple sequence alignments, suggest that one of two conserved glutamates, at positions 215 (209 in rat) and 337 (333 in rat), would act as the general base necessary to hydrolyze the thioacyl intermediate. While the latter is the general base in the rat Class 3 ALDH, site-specific mutagenesis indicates that Glu215 is the likely candidate for PpBADH, a result more typical of the Class 1 and 2 ALDH families. Finally, this study shows that hydride transfer is not rate limiting, lending further credence to the suggestion that PpBADH is more similar to the Class 1 and 2 ALDHs than it is to other Class 3 ALDHs.
恶臭假单胞菌的苯甲醛脱氢酶（PpBADH）属于3类醛脱氢酶（ALDH）家族。3类ALDH不同寻常之处在于它们通常是二聚体（而非四聚体），相对非特异性，并且同时利用NAD⁺和NADP⁺。迄今为止，已确定了3种3类ALDH的X射线结构，其中只有两种结合了辅因子，且均为NAD⁺形式。在此我们报道了PpBADH与NADP⁺及硫代酰基中间加合物的晶体结构。PpBADH的整体结构类似于ALDH超家族的大多数其他成员，且辅因子结合残基高度保守。相反，大鼠3类ALDH的辅因子结合模式与PpBADH及其他ALDH不同。这已根据大鼠酶的不同机制进行了解释。与PpBADH结构的比较以及多序列比对表明，在位置215（大鼠中为209）和337（大鼠中为3,33）的两个保守谷氨酸之一将作为水解硫代酰基中间体所需的通用碱。虽然后者是大鼠3类ALDH中的通用碱，但位点特异性诱变表明Glu215可能是PpBADH的候选者，这一结果更符合1类和2类ALDH家族的特征。最后，本研究表明氢化物转移不是限速步骤，这进一步支持了PpBADH与1类和2类ALDH比与其他3类ALDH更相似的观点。
