Physicochemical Characterization of a Thermostable Alcohol Dehydrogenase from Pyrobaculum aerophilum

Abstract

In this work we characterize an alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII). We have previously found that PyAeADHII has no activity when standard ADH substrates are used but is active when α-tetralone is used as substrate. Here, to gain insights into enzyme function, we screened several chemical libraries for enzymatic modulators using an assay employing α-tetralone. The results indicate that PyAeADHII activity in the presence of α-tetralone was inhibited by compounds such as flunarizine. We also examined metal coordination of the enzyme in solution by performing metal substitution of the enzyme-bound zinc (Zn2+) with cobalt. The solution-based absorption spectra for cobalt substituted PyAeADHII supports substitution at the structural Zn2+ site. To gain structural insight, we obtained the crystal structure of both wild-type and cobalt-substituted PyAeADHII at 1.75 Å and 2.20 Å resolution, respectively. The X-ray data confirmed one metal ion per monomer present only at the structural site with otherwise close conservation to other ADH enzymes. We next determined the co-crystal structure of the NADPH-bound form of the enzyme at 2.35 Å resolution to help define the active site region of the enzyme and this data shows close structural conservation with horse ADH, despite the lack of a catalytic Zn2+ ion in PyAeADHII. Modeling of α-tetralone into the NADPH bound structure suggests an arginine as a possible catalytic residue. The data presented here can yield a better understanding of alcohol dehydrogenases lacking the catalytic zinc as well as the structural features inherent to thermostable enzymes.

Expansion of the Catalytic Repertoire of Alcohol Dehydrogenases in Plant Metabolism**

“…Although electron density for the structural zinc ion was clearly observed in all Ti DPAS2 structures (the Cr DPAS structure lacked electron density for this region), surprisingly, the substrate pocket lacked electron density at the expected site of the catalytic zinc (Figure 2B, Figure S12). This has previously only been reported in a prokaryote ADH [14] . The lack of the catalytic zinc in DPAS is substantiated by the observation that two of the conserved catalytic zinc coordinating residues are mutated (His74Met and Cys168Ser, Figure S1).…”

Expansion of the Catalytic Repertoire of Alcohol Dehydrogenases in Plant Metabolism

“…Although electron density for the structural zinc ion was clearly observed in all Ti DPAS2 structures (the Cr DPAS structure lacked electron density for this region), surprisingly, the substrate pocket lacked electron density at the expected site of the catalytic zinc (Figure 2B, Figure S11). This has previously only been reported in a prokaryote [14] . The lack of the catalytic zinc in DPAS is substantiated by the observation that two of the conserved catalytic zinc coordinating residues are mutated (His74Met and Cys168Ser, Figure S1).…”

Decarboxylation of Pyruvate to Acetaldehyde for Ethanol Production by Hyperthermophiles

“…Many different ADHs have been characterized from various thermophilic and hyperthermophilic bacteria and archaea, with a majority of them being NAD(P)-dependent. Some of the more recently characterized hyper/thermophilic ADHs are those from P. furiosus [ 53 , 54 ], Thermococcus hydrothermalis [ 55 ], Thermococcus kodakarensis [ 56 , 57 , 58 ], Thermococcus sibiricus [ 59 , 60 ], Thermococcus guaymasensis [ 42 ], Sulfolobus acidocaldarius [ 61 ], Thermococcus strain ES1 [ 62 ], Aeropyrum pernix [ 63 ], Thermotoga hypogea [ 64 ], and Pyrobaculum aerophilum [ 65 ].…”