Crystal Structure of Aldehyde Dehydrogenase 16 Reveals Trans-Hierarchical Structural Similarity and a New Dimer

Abstract

The aldehyde dehydrogenase (ALDH) superfamily is a vast group of enzymes that catalyze the NAD + -dependent oxidation of aldehydes to carboxylic acids. ALDH16 is perhaps the most enigmatic member of the superfamily, owing to its extra C-terminal domain of unknown function and the absence of the essential catalytic cysteine residue in certain non-bacterial ALDH16 sequences. Herein we report the first production of recombinant ALDH16, the first biochemical characterization of ALDH16, and the first crystal structure of ALDH16. Recombinant expression systems were generated for the bacterial ALDH16 from Loktanella sp. and human ALDH16A1. Four high resolution crystal structures of Loktanella ALDH16 were determined. Loktanella ALDH16 is found to be a bona fide enzyme, exhibiting NAD + -binding, ALDH activity, and esterase activity. In contrast, human ALDH16A1 apparently lacks measurable aldehyde oxidation activity, suggesting it is a pseudoenzyme, consistent with the absence of the catalytic Cys in its sequence. The fold of ALDH16 comprises three domains: NAD + -binding, catalytic, and Cterminal. The latter is unique to ALDH16 and features a Rossmann fold connected to a protruding β-flap. The tertiary structural interactions of the C-terminal domain mimic the quaternary structural interactions of the classic ALDH superfamily dimer, a phenomenon we call "transhierarchical structural similarity". ALDH16 forms a unique dimer in solution, which mimics the classic ALDH superfamily dimer-of-dimers tetramer. Small-angle X-ray scattering shows that human ALDH16A1 has the same dimeric structure and fold as Loktanella ALDH16. We suggest the Loktanella ALDH16 structure may be considered to be the archetype of the ALDH16 family.

Insights into Aldehyde Dehydrogenase Enzymes: A Structural Perspective

“…Interestingly these enzymes utilise the same active site residues to carry out this function as dehydrogenase catalysis, however esterase activity does not require the addition of the NAD(P) cofactor (Koppaka et al, 2012). The so-called "pseudoenzyme" human ALDH16 lacks this catalytic Cys residue, resulting in absence of catalytic activity, proposed to function as a binding protein (Liu and Tanner, 2019).…”

“…Of these, glycines and prolines were abundant (7 and 2, respectively), likely highlighting critical chain-bending points due to the antihelical and beta-sheet potentials of these residues (Perozich et al, 1999b). The catalytic Cys is conserved in all structures which harbor catalytic activity, the above mentioned ALDH16 being a prime example of a non-catalytic member (Liu and Tanner, 2019). In addition, two other important conserved residues associated with catalysis, lysine (Lys192) and glutamic acid (Glu268) (human numbering), are evident across the ALDHs.…”

Structure and mechanism of piperideine-6-carboxylate dehydrogenase fromStreptomyces clavuligerus

“…The homodimers can further assemble into homotetramers or homohexamers (Koppaka et al, 2012;Luo et al, 2013). Other more unusual oligomeric arrangements have also been observed (Liu & Tanner, 2019). P6CDH crystallizes with a homodimer in the asymmetric unit, but the crystal lattice shows tight interaction with a second dimer, indicating that the oligomeric composition of P6CDH may in fact be a dimer-of-dimers tetramer.…”

Expression, purification and crystallization of the novel Xenopus tropicalis ALDH16B1, a homologue of human ALDH16A1

“…The members of the ALDH superfamily share a similar quaternary structure which comprises an NAD(P) + binding domain, a catalytic domain that contains the critical cysteine (Cys) residue, and a smaller oligomerization domain. Although the most common oligomeric state of ALDHs is tetrameric, both dimeric and hexameric biological assemblies have also been reported [8]. Among the nineteen members of the ALDH superfamily, the ALDH16 family is the least characterized.…”

“…Among the nineteen members of the ALDH superfamily, the ALDH16 family is the least characterized. The crystal structure of bacterial ALDH16 was recently reported [8]. Structurally, the members of this family contain an extra C-terminal domain, the role of which is poorly understood.…”