Mechanistic and Structural Studies of Apoform, Binary, and Ternary Complexes of the Arabidopsis Alkenal Double Bond Reductase At5g16970

Abstract

In this study, we determined the crystal structures of the apoform, binary, and ternary complexes of the Arabidopsis alkenal double bond reductase encoded by At5g16970. This protein, one of 11 homologues in Arabidopsis thaliana, is most closely related to the Pinus taeda phenylpropenal double bond reductase, involved in, for example, heartwood formation. Both enzymes also have essential roles in plant defense, and can function by catalyzing the reduction of the 7-8-double bond of phenylpropanal substrates, such as p-coumaryl and coniferyl aldehydes in vitro. At5g16970 is also capable of reducing toxic substrates with the same alkenal functionality, such as 4-hydroxy-(2E)-nonenal. The overall fold of At5g16970 is similar to that of the zinc-independent medium chain dehydrogenase/reductase superfamily, the members of which have two domains and are dimeric in nature, i.e. in contrast to their original classification as being zinc-containing oxidoreductases. As provisionally anticipated from the kinetic data, the shape of the binding pocket can readily accommodate p-coumaryl aldehyde, coniferyl aldehyde, 4-hydroxy-(2E)-nonenal, and 2-alkenals. However, the enzyme kinetic data among these potential substrates differ, favoring p-coumaryl aldehyde. Tyr-260 is provisionally proposed to function as a general acid/base for hydride transfer. A catalytic mechanism for this reduction, and its applicability to related important detoxification mammalian proteins, is also proposed.

Different Routes for Conifer- and Sinapaldehyde and Higher Saccharification upon Deficiency in the Dehydrogenase CAD1

“…The accumulation of dihydroferulic acid hexosides 5 and 12 can possibly be explained by the activity of an alkenal DBR. The activities of an Arabidopsis DBR reducing the C7-C8 double bond of p-coumaraldehyde and coniferaldehyde, and an apple (Malus 3 domestica) DBR reducing the double bond in p-coumaroyl-CoA and feruloyl-CoA, have been shown by enzymatic assays (Youn et al, 2006b;Ibdah et al, 2014). Vanillic acid hexoside 13 also accumulated in the CAD1-deficient poplars (Table V).…”

“…Thus, for this mechanism to operate, a prior double bond reduction of sinapic acid to dihydrosinapic acid (or from sinapaldehyde to dihydrosinapaldehyde followed by its oxidation to dihydrosinapic acid) is necessary, after which dihydrosinapic acid is hydroxylated to syringyl lactic acid. As stated above, DBR enzymes from Arabidopsis and apple reducing the C7-C8 double bond of phenylpropanoids have been described (Youn et al, 2006b;Ibdah et al, 2014).…”

“…06, 208.04, 179.07, 164.05, and 149.02 were reminiscent of a sinapic acid-derived moiety (Widhalm and Dudareva, 2015). DBR has been found to reduce the C7-C8 double bond of p-coumaraldehyde and coniferaldehyde in Arabidopsis (Youn et al, 2006b), but it also might reduce the corresponding hydroxycinnamoyl-CoAs (Ibdah et al, 2014). The functions of a heterotrimeric C4H/C3H protein complex in the hydroxylation of p-coumaryl shikimate toward caffeoyl shikimate and a similar heterodimeric C4H/C3H protein complex that is able to convert p-coumaric acid directly into caffeic acid in poplar have been described by Chen et al (2011).…”

Three-dimensional Structure and Enzymatic Function of Proapoptotic Human p53-inducible Quinone Oxidoreductase PIG3

“…In contrast, in bovine -crystallin, which shows minimal QOR activity, the corresponding residue is a His, suggesting that the Tyr is essential in the kinetic mechanism (51). In addition, mutagenesis and/or structural data support the participation of a Tyr in the mechanism of the related, Zn 2ϩ -lacking MDRs (Table 2) enoyl reductase (39,47), Arabidopsis NADP-dependent oxidoreductase P1, and leukotriene B 4 12-hydroxydehydrogenase (45,46). Thus, it was appropriate to propose a catalytic role for PIG3 Tyr-51, the only Tyr residue in the active-site cavity, further supported by a docking model (Fig.…”

“…Site-directed Mutagenesis of the Active Site Tyr-In several Zn 2ϩ -independent MDRs, evidence supports a Tyr residue acting as an acid/base catalyst (39,(45)(46)(47). The crystallographic structures suggest that Tyr-51 in PIG3 and Tyr-59 in -crystallin are candidates to perform this catalytic function.…”