In the human pathogen Pseudomonas aeruginosa, the
NAD(P)(+)-dependent
betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating
carbon and
nitrogen from
choline or
choline precursors--abundant at
infection sites--and producing
glycine betaine and
NADPH, potentially protective against the high-osmolarity and oxidative stresses prevalent in the infected tissues. Disruption of the PaBADH gene negatively affects the growth of bacteria, suggesting that this
enzyme could be a target for
antibiotic design. PaBADH is one of the few ALDHs that efficiently use
NADP(+) and one of the even fewer that require K(+)
ions for stability. Crystals of PaBADH were obtained under aerobic conditions in the presence of
2-mercaptoethanol,
glycerol,
NADP(+) and K(+)
ions. The three-dimensional structure was determined at 2.1-A resolution. The catalytic
cysteine (C286, corresponding to C302 of ALDH2) is oxidized to
sulfenic acid or forms a mixed
disulfide with
2-mercaptoethanol. The glutamyl residue involved in the deacylation step (E252, corresponding to E268 of ALDH2) is in two conformations, suggesting a
proton relay system formed by two well-conserved residues (E464 and K162, corresponding to E476 and K178, respectively, of ALDH2) that connects E252 with the bulk water. In some active sites, a bound
glycerol molecule mimics the thiohemiacetal intermediate; its
hydroxyl oxygen is
hydrogen bonded to the
nitrogen of the
amide groups of the side chain of the conserved N153 (N169 of ALDH2) and those of the main chain of C286, which form the "oxyanion hole." The
nicotinamide moiety of the
nucleotide is not observed in the crystal, and the
adenine moiety binds in the usual way. A
salt bridge between E179 (E195 of ALDH2) and R40 (E53 of ALDH2) moves the carboxylate group of the former away from the 2'-phosphate of the
NADP(+), thus avoiding steric clashes and/or electrostatic repulsion between the two groups. Finally, the crystal shows two K(+) binding sites per subunit. One is in an intrasubunit cavity that we found to be present in all known ALDH structures. The other--not described before for any ALDH but most likely present in most of them--is located in between the dimeric unit, helping structure a region involved in
coenzyme binding and catalysis. This may explain the effects of K(+)
ions on the activity and stability of PaBADH.