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.
González-Segura
Lilian
L
Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, México DF, México.
Mújica-Jiménez
Carlos
C
Muñoz-Clares
Rosario A
RA
D001152:Arsenicals / Q000378:metabolism*; D018053:Arsenites / Q000378:metabolism*; D050728:Betaine-Aldehyde Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D055162:Biocatalysis; D003545:Cysteine / Q000378:metabolism*; D004112:Dimercaprol / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D001152:Arsenicals; D018053:Arsenites; D011994:Recombinant Proteins; D004112:Dimercaprol; C029341:oxophenylarsine; D050728:Betaine-Aldehyde Dehydrogenase; D003545:Cysteine; C015001:arsenite