Structure-based mutational studies of substrate inhibition of betaine aldehyde dehydrogenase BetB from Staphylococcus aureus.

Inhibition of enzyme activity by high concentrations of substrate and/or cofactor is a general phenomenon demonstrated in many enzymes, including aldehyde dehydrogenases. Here we show that the uncharacterized protein BetB (SA2613) from Staphylococcus aureus is a highly specific betaine aldehyde dehydrogenase, which exhibits substrate inhibition at concentrations of betaine aldehyde as low as 0.15 mM. In contrast, the aldehyde dehydrogenase YdcW from Escherichia coli, which is also active against betaine aldehyde, shows no inhibition by this substrate. Using the crystal structures of BetB and YdcW, we performed a structure-based mutational analysis of BetB and introduced the YdcW residues into the BetB active site. From a total of 32 mutations, those in five residues located in the substrate binding pocket (Val288, Ser290, His448, Tyr450, and Trp456) greatly reduced the substrate inhibition of BetB, whereas the double mutant protein H448F/Y450L demonstrated a complete loss of substrate inhibition. Substrate inhibition was also reduced by mutations of the semiconserved Gly234 (to Ser, Thr, or Ala) located in the BetB NAD(+) binding site, suggesting some cooperativity between the cofactor and substrate binding sites. Substrate docking analysis of the BetB and YdcW active sites revealed that the wild-type BetB can bind betaine aldehyde in both productive and nonproductive conformations, whereas only the productive binding mode can be modeled in the active sites of YdcW and the BetB mutant proteins with reduced substrate inhibition. Thus, our results suggest that the molecular mechanism of substrate inhibition of BetB is associated with the nonproductive binding of betaine aldehyde.

The disulfiram metabolites S-methyl-N,N-diethyldithiocarbamoyl sulfoxide and S-methyl-N,N-diethylthiocarbamoyl sulfone irreversibly inactivate betaine aldehyde dehydrogenase from Pseudomonas aeruginosa, both in vitro and in situ, and arrest bacterial growth.

Betaine aldehyde dehydrogenase from the human opportunistic pathogen Pseudomonas aeruginosa (PaBADH) catalyzes the irreversible, NAD(P)(+)-dependent oxidation of betaine aldehyde, producing glycine betaine, an osmoprotectant. PaBADH participates in the catabolism of choline and likely in the defense against the osmotic and oxidative stresses to which the bacterium is exposed when infecting human tissues. Given that choline or choline precursors are abundant in infected tissues, PaBADH is a potential drug target because its inhibition will lead to the build up of the toxic betaine aldehyde inside bacterial cells. We tested the thiol reagents, disulfiram (DSF) and five DSF metabolites-diethyldithiocarbamic acid (DDC), S-methyl-N,N-diethyldithiocarbamoyl sulfoxide (MeDDTC-SO) and sulfone (MeDDTC-SO(2)), and S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO) and sulfone (MeDTC-SO(2))-as inhibitors of PaBADH and P. aeruginosa growth. As in vitro PaBADH inhibitors, their order of potency was: MeDDTC-SO(2)>DSF>MeDTC-SO(2)>MeDDTC-SO>MeDTC-SO. DDC did not inactivate the enzyme. PaBADH inactivation by DSF metabolites (i) was not affected by NAD(P)(+), (ii) could not be reverted by dithiothreitol, and (iii) did not affect the quaternary structure of the enzyme. Of the DSF metabolites tested, MeDTC-SO(2) and MeDDTC-SO produced significant in situ PaBADH inactivation and arrest of P. aeruginosa growth in choline containing media, in which the expression of PaBADH is induced. They had no effect in media lacking choline, indicating that PaBADH is their main intracellular target, and that arrest of growth is due to accumulation of betaine aldehyde. The in vitro and in situ kinetics of enzyme inactivation by these two compounds were very similar, indicating no restriction on their uptake by the cells. MeDDTC-SO(2) and DSF have no inhibitory effects in situ, probably because their high reactivity towards intracellular nonessential thiols causes their depletion. Our results support that PaBADH is a promising target to treat P. aeruginosa infections, and that some DSF metabolites might be of help in this aim.

Kinetic and structural features of betaine aldehyde dehydrogenases: mechanistic and regulatory implications.

The betaine aldehyde dehydrogenases (BADH; EC 1.2.1.8) are so-called because they catalyze the irreversible NAD(P)(+)-dependent oxidation of betaine aldehyde to glycine betaine, which may function as (i) a very efficient osmoprotectant accumulated by both prokaryotic and eukaryotic organisms to cope with osmotic stress, (ii) a metabolic intermediate in the catabolism of choline in some bacteria such as the pathogen Pseudomonas aeruginosa, or (iii) a methyl donor for methionine synthesis. BADH enzymes can also use as substrates aminoaldehydes and other quaternary ammonium and tertiary sulfonium compounds, thereby participating in polyamine catabolism and in the synthesis of gamma-aminobutyrate, carnitine, and 3-dimethylsulfoniopropionate. This review deals with what is known about the kinetics and structural properties of these enzymes, stressing those properties that have only been found in them and not in other aldehyde dehydrogenases, and discussing their mechanistic and regulatory implications.

Reaction of the catalytic cysteine of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa with arsenite-BAL and phenylarsine oxide.

Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)(+) to NAD(P)H. In the opportunistic pathogen Pseudomonas aeruginosa, this enzyme (PaBADH) could be an antimicrobial target. Several aldehyde dehydrogenases (ALDHs) are inactivated by arsenite in the presence of a low molecular thiol, a finding that was interpreted as a demonstration of the existence of vicinal thiols in these enzymes. As part of our studies on the susceptibility to chemical modification of the catalytic cysteine (C286) of PaBADH, we treated the enzyme with two arsenical reagents widely used to inhibit enzymes that have vicinal thiols: sodium m-arsenite plus 2,3-dimercaptopropanol (arsenite-BAL) and phenylarsine oxide (PAO). Here we report that they readily and reversibly inactivate PaBADH, even though the four cysteine residues of this enzyme (C286, C353, C377, and C439) are far from each other in the three-dimensional structure. Modification of PaBADH by both reagents was reversible by an excess of a dithiol (dithiothreitol), but only the PAO-modified enzyme could be reactivated by a monothiol (2-mercaptoethanol). C286 is the reactive residue as indicated by the following findings: (i) betaine aldehyde and NADP(+) afforded full protection against enzyme inactivation; (ii) the mutant proteins C353A, C377A, and C439A showed similar inactivation kinetics that the wild-type enzyme, and (iii) pretreatment of PaBADH with arsenite-BAL prevented irreversible inactivation by N-ethylmaleimide. Our results confirm previous findings on other ALDHs, and indicate that these vicinal thiol-specific reagents readily react with certain monothiols, such as the one of the catalytic cysteinyl residue of ALDHs. As arsenicals are being recently used to treat certain cancers, human ALDHs, even those not having conformationally vicinal thiols, may be unsuspected targets in these treatments.