NADP-Dependent Aldehyde Dehydrogenase from ArchaeonPyrobaculum sp.1860: Structural and Functional Features

Abstract

We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. In vitro, AlDHPyr1147 catalyzes the irreversible oxidation of short aliphatic aldehydes at 60–85°С, and the affinity of AlDHPyr1147 to the NADP+ at 60°С is comparable to that for mesophilic analogues at 25°С. We determined the structures of the apo form of AlDHPyr1147 (3.04 Å resolution), three binary complexes with the coenzyme (1.90, 2.06, and 2.19 Å), and the ternary complex with the coenzyme and isobutyraldehyde as a substrate (2.66 Å). The nicotinamide moiety of the coenzyme is disordered in two binary complexes, while it is ordered in the ternary complex, as well as in the binary complex obtained after additional soaking with the substrate. AlDHPyr1147 structures demonstrate the strengthening of the dimeric contact (as compared with the analogues) and the concerted conformational flexibility of catalytic Cys287 and Glu253, as well as Leu254 and the nicotinamide moiety of the coenzyme. A comparison of the active sites of AlDHPyr1147 and dehydrogenases characterized earlier suggests that proton relay systems, which were previously proposed for dehydrogenases of this family, are blocked in AlDHPyr1147, and the proton release in the latter can occur through the substrate channel.

NADP(H)-dependent biocatalysis without adding NADP(H)

“…S1 A and B ) ( 14 , 19 – 22 ). Other proteins that copurify with bound NAD(H) ( 23 – 32 ) and NADP(H) ( 33 – 39 ) include the nicotinoproteins ( 28 – 32 , 37 , 38 , 40 ) that apparently use NAD(P)(H) as a permanently bound prosthetic group (instead of an exchangeable cofactor) to perform a variety of reactions. The limited examples of NAD(P)(H)-dependent dehydrogenases copurifying with nicotinamide cofactors ( 23 , 24 , 32 – 35 ) suggest that extremely tight cofactor binding may be unusual.…”

An NAD-Specific 6-Hydroxy-3-Succinoyl-Semialdehyde-Pyridine Dehydrogenase from Nicotine-Degrading Agrobacterium tumefaciens Strain S33

“…S3B). According to the known structures of various aldehyde dehydrogenases ( 33 – 36 ), the sequence close to the C terminus of some aldehyde dehydrogenases is related to the dimer formation. The His tag at the C terminus affects the binding of the dimer, resulting in instability and low activity of the enzyme.…”

Crystal structure of thermostable acetaldehyde dehydrogenase from the hyperthermophilic archaeon Sulfolobus tokodaii

“…The active-site conformation of ALDHSt is homologous to that of ALDHPs (Bezsudnova et al, 2016). In the apo form of ALDHPs, a hydrogen bond was observed between Glu253 and Gly255 (Bezsudnova et al, 2016). However, no hydrogen bond was observed between Glu240 and Gly242 (corresponding to Glu253 and Gly255, respectively, in ALDHPs).…”

“…3c). A comparison of previously characterized ALDHPs active sites (Liu et al, 2013;Bezsudnova et al, 2016) suggests that the catalytic side chain of Cys274 is directed away from the NADP-binding site (Fig. 3c) and attacks the aldehyde group of the substrate in the first step of the enzyme reaction.…”

“…These results indicate that the structure of ALDHSt in this study is a holo form complexed with NADP. Focusing on the cofactor-binding site, the catalytic side chains of Glu253 and Cys287 in ALDHPs (Bezsudnova et al, 2016) were found to be well conserved in the corresponding residues, Glu240 and Cys274, in ALDHSt (Fig. 3c).…”