X-ray structure of the quinoprotein ethanol dehydrogenase from Pseudomonasaeruginosa: Basis of substrate specificity

The homodimeric enzyme form of quinoprotein ethanol dehydrogenase from Pseu domonas aeruginosa ATCC 17933 crystallizes readily with the space group R3. The X-ray structure was solved at 2.6 Angstrom resolution by molecular rep lacement. Aside from differences in some loops, the folding of the enzyme is very sim ilar to the large subunit of the quinoprotein methanol dehydrogenases from Methylobacterium extorquens or Methylophilus W3A1. Eight W-shaped beta-shee t motifs are arranged circularly in a propeller-like fashion forming a disk -shaped superbarrel. No electron density for a small subunit like that in m ethanol dehydrogenase could be found. The prosthetic group is located in th e centre of the superbarrel and is coordinated to a calcium ion. Most amino acid residues found in close contact with the prosthetic group pyrroloquin oline quinone and the Ca2+ are conserved between the quinoprotein ethanol d ehydrogenase structure and that of the methanol dehydrogenases. The main di fferences in the active-site region are a bulky tryptophan residue in the a ctive-site cavity of methanol dehydrogenase, which is replaced by a phenyla lanine and a leucine side-chain in the ethanol dehydrogenase structure and a leucine residue right above the pyrrolquinoline quinone group in methanol dehydrogenase which is replaced by a tryptophan side-chain. Both amino aci d exchanges appear to have an important influence, causing different substr ate specificities of these otherwise very similar enzymes. In addition to t he Ca2+ in the active-site cavity found also in methanol dehydrogenase, eth anol dehydrogenase contains a second Ca2+-binding site at the N terminus, w hich contributes to the stability of the native enzyme. (C) 2000 Academic P ress.