Molecular Genetic and Crystal Structural Analysis of 1-(4-Hydroxyphenyl)-Ethanol Dehydrogenase from 'Aromatoleum aromaticum' EbN1.
The dehydrogenation of 1-(4-hydroxyphenyl)-ethanol to 4-hydroxyacetophenone represents the second reaction step during anaerobic degradation of p-ethylphenol in the denitrifying bacterium 'Aromatoleum aromaticum' EbN1. Previous proteogenomic studies identified two different proteins (ChnA and EbA309) as possible candidates for catalyzing this reaction [Wöhlbrand et al: J Bacteriol 2008;190:5699-5709]. Physiological-molecular characterization of newly generated unmarked in-frame deletion and complementation mutants allowed defining ChnA (renamed here as Hped) as the enzyme responsible for 1-(4-hydroxyphenyl)-ethanol oxidation. Hped [1-(4-hydroxyphenyl)-ethanol dehydrogenase] belongs to the 'classical' family within the short-chain alcohol dehydrogenase/reductase (SDR) superfamily. Hped was overproduced in Escherichia coli, purified and crystallized. The X-ray structures of the apo- and NAD(+)-soaked form were resolved at 1.5 and 1.1 Å, respectively, and revealed Hped as a typical homotetrameric SDR. Modeling of the substrate 4-hydroxyacetophenone (reductive direction of Hped) into the active site revealed the structural determinants of the strict (R)-specificity of Hped (Phe(187)), contrasting the (S)-specificity of previously reported 1-phenylethanol dehydrogenase (Ped; Tyr(93)) from strain EbN1 [Höffken et al: Biochemistry 2006;45:82-93].
Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl von Ossietzky University Oldenburg, Oldenburg, Germany.
Imke
I
Büsing
H Wolfgang
HW
Höffken
Michael
M
Breuer
Lars
L
Wöhlbrand
Bernhard
B
Hauer
Ralf
R
Rabus
D000098:Acetophenones / Q000737:chemistry / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005285:Fermentation; D062105:Molecular Docking Simulation / Q000379:methods; D009154:Mutation; D010626:Phenylethyl Alcohol / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D043362:Rhodocyclaceae / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D020539:Sequence Analysis, Protein
D000098:Acetophenones; D011994:Recombinant Proteins; C011867:4-hydroxyphenylethanol; D000426:Alcohol Dehydrogenase; C031335:4-hydroxyacetophenone; D010626:Phenylethyl Alcohol