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

Dinh, Tung; Rahn, K. Troy; Phillips, Robert S.

doi:10.1002/prot.26339

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

Journal Article · Sat Apr 09 00:00:00 EDT 2022 · Proteins

DOI:https://doi.org/10.1002/prot.26339· OSTI ID:1896916

Dinh, Tung ^[1]; Rahn, K. Troy ^[1]; ^[2]

Department of Chemistry University of Georgia Athens Georgia USA
Department of Chemistry University of Georgia Athens Georgia USA, Department of Biochemistry and Molecular Biology University of Georgia Athens Georgia USA, Center for Metalloenzyme Studies University of Georgia Athens Georgia USA

Abstract

Three‐dimensional structures of I86A and C295A mutant secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter pseudoethanolicus were determined by x‐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′‐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.

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Sponsoring Organization:: USDOE

Grant/Contract Number:: W-31-109-ENG-3

OSTI ID:: 1896916

Alternate ID(s):: OSTI ID: 1862419
OSTI ID: 1877196

Journal Information:: Proteins, Journal Name: Proteins Journal Issue: 8 Vol. 90; ISSN 0887-3585

Publisher:: Wiley Blackwell (John Wiley & Sons)Copyright Statement

Country of Publication:: United States

Language:: English

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