Crystallographic snapshots of ternary complexes of thermophilic secondary alcohol dehydrogenase from Thermoanaerobacter pseudoethanolicus reveal the dynamics of ligand exchange and the proton relay network

Abstract

Three-dimensional structures of I86A and C295A mutant secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter pseudoethanolicus were determined byx-ray crystallography. The tetrameric structure of C295A-SADH soaked with NADP + and dimethyl sulfoxide (DMSO) was determined to 1.85 Å with an R free of 0.225.DMSO is bound to the tetrahedral zinc in each subunit, with ligands from SG of Cys-37, NE2 of His-59, and OD2 of Asp-150. The nicotinamide ring of NADP is hydrogen-bonded to the N of Ala-295 and the O of Val-265 and Gly-293. The O of DMSO is connected to a network of hydrogen bonds with OG of Ser-39, the 3 0 -OH of NADP, and ND1 of His-42. The structure of I86A-SADH soaked with 2-pentanol and NADP + contains (R)-2-pentanol bound in each subunit, ligated to the tetrahedral zinc, and connected to the proton relay network. The structure of I86A-SADH soaked with 3-methylcyclohexanol and NADP + has alcohol bound in three subunits.Two of the sites have the alcohol ligated to the zinc in an axial position, with OE2 of Glu-60 in the other axial position of a trigonal bipyramidal complex. One site has 3-methylcyclohexanol bound noncovalently, with the zinc in an inverted tetrahedral geometry with Glu-60. The fourth site also has the zinc in a trigonal bipyramidal complex with axial Glu-60 and water ligands. These structures demonstrate that ligand exchange of SADH involves pentacoordinate and inverted zinc complexes with Glu-60. Furthermore, we see a network of hydrogen bonds connecting the substrate oxygen to the external solvent that is likely to play a role in the mechanism of SADH.enzyme mechanism, pentacoordinate zinc, stereospecificity, tetrahedral zinc, zinc complex | INTRODUCTIONAlcohol dehydrogenases are valuable catalysts for the asymmetric reduction of ketones to produce chiral alcohols. The thermostable secondary alcohol dehydrogenases (SADH) from Thermoanaerobacter brockii and Thermoanaerobacter (pseudo) ethanolicus have been extensively used for this purpose. [1][2][3] The enzymes from these two bacterial sources have been shown recently to have an identical amino acid sequence. 2 This enzyme is a member of the medium-chain dehydrogenase/reductases (MDRs) family. Similar to other MDRs, each chain of SADH contains 352 amino acid residues. However, it shares only 27% sequence identity with the more widely studied horse liver alcohol dehydrogenase (HLADH). Because of its potential application in chiral syntheses, there have been numerous studies to broaden the

The metal cofactor: stationary or mobile?

“…The mechanistic role of Zn(II) has been studied in detail in other ADH enzymes of the MDR family, such as Saccharomyces cerevisiae ADH1 ( Sc ADH1) (Guntupalli et al 2021 ; Plapp et al 2016 ), horse liver ADH1E (HlADH) (Plapp et al 2017 ), and Thermoanaerobacter pseudoethanolicus ADH ( Tp ADH)(Dinh et al 2022 ). In Sc ADH1, the Zn(II) is tetrahedrally coordinated and the two structures observed in the subunits indicate a double inversion of the Zn(II) during coordination; first, the coordinated water is released and replaced by glutamate; subsequently, the second inversion leads to the coordination of the substrate alcohol in the position previously taken by water (Fig.…”