Modulation of the reactivity of the essential cysteine residue of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

2002 ◽  
Vol 361 (3) ◽  
pp. 577-585 ◽  
Author(s):  
Lilian GONZÁLEZ-SEGURA ◽  
Roberto VELASCO-GARCÍA ◽  
Rosario A. MUÑOZ-CLARES
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Aldehyde Dehydrogenase ◽  
Ph Dependence ◽  
Catalytic Efficiency ◽  
Opportunistic Pathogen ◽  
Order Rate Constant ◽  
Cysteine Residue ◽  
Ion Pair ◽  
Betaine Aldehyde Dehydrogenase ◽  
Betaine Aldehyde

Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible NAD(P)+-dependent oxidation of betaine aldehyde to glycine betaine. In the human opportunistic pathogen Pseudomonas aeruginosa this reaction is an obligatory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. As with every aldehyde dehydrogenase studied so far, BADH possesses an essential cysteine residue involved in the formation of the intermediate thiohemiacetal with the aldehyde substrate. We report here that the chemical modification of this residue is conveniently measured by the loss in enzyme activity, which allowed us to explore its reactivity in a pH range around neutrality. The pH dependence of the observed second-order rate constant of BADH inactivation by methyl methanethiosulphonate (MMTS) suggests that at low pH values the essential cysteine residue exists as thiolate by the formation of an ion pair with a positively charged residue. The estimated macroscopic pK values are 8.6 and 4.0 for the free and ion-pair-forming thiolate respectively. The reactivity towards MMTS of both thiolate forms is notably lower than that of model compounds of similar pK, suggesting a considerable steric inhibition by the structure of the protein. Binding of the dinucleotides rapidly induced a significant and transitory increment of thiolate reactivity, followed by a relatively slow change to an almost unreactive form. Thus it seems that to gain protection against oxidation without compromising catalytic efficiency, BADH from P. aeruginosa has evolved a complex and previously undescribed mechanism, involving several conformational rearrangements of the active site, to suit the reactivity of the essential thiol to the availability of coenzyme and substrate.

scholarly journals Rapid Purification and Properties of Betaine Aldehyde Dehydrogenase from Pseudomonas aeruginosa

1999 ◽  
Vol 181 (4) ◽  
pp. 1292-1300 ◽  
Author(s):  
Roberto Velasco-García ◽  
Carlos Mújica-Jiménez ◽  
Guillermo Mendoza-Hernández ◽  
Rosario A. Muñoz-Clares
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Aldehyde Dehydrogenase ◽  
Ph Dependence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Betaine Aldehyde Dehydrogenase ◽  
Betaine Aldehyde ◽  
Cell Extracts ◽  
Rapid Purification

ABSTRACT Betaine aldehyde dehydrogenase (BADH) (EC 1.2.1.8 ) catalyzes the last, irreversible step in the synthesis of the osmoprotectant glycine betaine from choline. In Pseudomonas aeruginosa this reaction is also an obligatory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. We present here a method for the rapid purification to homogeneity of this enzyme by the use of ion-exchange and affinity chromatographies on 2′,5′-ADP–Sepharose, which results in a high yield of pure enzyme with a specific activity at 30°C and pH 7.4 of 74.5 U/mg of protein. Analytical ultracentrifugation, gel filtration, chemical cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggest that BADH from P. aeruginosa is a homodimer with 61-kDa subunits. The amino acid composition and the N-terminal sequence of 21 amino acid residues showed significant similarity with those of the enzymes from Xanthomonas translucens andEscherichia coli. Neither BADH activity nor BADH protein was found in cell extracts from bacteria grown in the absence of choline. In contrast to other BADHs studied to date, thePseudomonas enzyme cannot use positively charged aldehydes other than betaine aldehyde as substrates. The oxidation reaction has an activation energy of 39.8 kJ mol−1. The pH dependence of the velocity indicated an optimum at pH 8.0 to 8.5 and the existence of two ionizable groups with macroscopic pK values of 7.0 ± 0.1 and 9.7 ± 0.1 involved in catalysis and/or binding of substrates. The enzyme is inactivated at 40°C, but activity is regained when the heated enzyme is cooled to 30°C or lower. At the optimum pH of 8.0, the enzyme is inactivated by dilution, but it is stable at pH 6.5 even at very low concentrations. Also, P. aeruginosa BADH activity is rapidly lost on removal of K+. In all cases studied, inactivation involves a biphasic process, which was dependent on the enzyme concentration only in the case of inactivation by dilution. NADP+ considerably protected the enzyme against these inactivating conditions.

scholarly journals Steady-state kinetic mechanism of the NADP+- and NAD+-dependent reactions catalysed by betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

2000 ◽  
Vol 352 (3) ◽  
pp. 675-683 ◽  
Author(s):  
Roberto VELASCO-GARCÍA ◽  
Lilian GONZÁLEZ-SEGURA ◽  
Rosario A. MUÑOZ-CLARES
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Betaine Aldehyde Dehydrogenase ◽  
Betaine Aldehyde ◽  
Metabolic Role ◽  
Steady State Kinetic ◽  
State Kinetic

Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)+ to NADP(H). In Pseudomonas aeruginosa this reaction is a compulsory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. The kinetic mechanisms of the NAD+- and NADP+-dependent reactions were examined by steady-state kinetic methods and by dinucleotide binding experiments. The double-reciprocal patterns obtained for initial velocity with NAD(P)+ and for product and dead-end inhibition establish that both mechanisms are steady-state random. However, quantitative analysis of the inhibitions, and comparison with binding data, suggest a preferred route of addition of substrates and release of products in which NAD(P)+ binds first and NAD(P)H leaves last, particularly in the NADP+-dependent reaction. Abortive binding of the dinucleotides, or their analogue ADP, in the betaine aldehyde site was inferred from total substrate inhibition by the dinucleotides, and parabolic inhibition by NADH and ADP. A weak partial uncompetitive substrate inhibition by the aldehyde was observed only in the NADP+-dependent reaction. The kinetics of P. aeruginosa BADH is very similar to that of glucose-6-phosphate dehydrogenase, suggesting that both enzymes fulfil a similar amphibolic metabolic role when the bacteria grow in choline and when they grow in glucose.

Site-directed mutagenesis and homology modeling indicate an important role of cysteine 439 in the stability of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

Biochimie ◽  
2005 ◽  
Vol 87 (12) ◽  
pp. 1056-1064 ◽  
Author(s):  
Lilian González-Segura ◽  
Roberto Velasco-García ◽  
Enrique Rudiño-Piñera ◽  
Carlos Mújica-Jiménez ◽  
Rosario A. Muñoz-Clares
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Homology Modeling ◽  
Aldehyde Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Betaine Aldehyde Dehydrogenase ◽  
Betaine Aldehyde ◽  
The Stability

Betaine aldehyde dehydrogenase from Pseudomonas aeruginosa: cloning, over-expression in Escherichia coli, and regulation by choline and salt

2005 ◽  
Vol 185 (1) ◽  
pp. 14-22 ◽  
Author(s):  
Roberto Velasco-García ◽  
Miguel Angel Villalobos ◽  
Miguel A. Ramírez-Romero ◽  
Carlos Mújica-Jiménez ◽  
Gabriel Iturriaga ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Aldehyde Dehydrogenase ◽  
Betaine Aldehyde Dehydrogenase ◽  
Over Expression ◽  
Betaine Aldehyde ◽  
Expression In Escherichia Coli

Novel NADPH–cysteine covalent adduct found in the active site of an aldehyde dehydrogenase

2011 ◽  
Vol 439 (3) ◽  
pp. 443-455 ◽  
Author(s):  
Ángel G. Díaz-Sánchez ◽  
Lilian González-Segura ◽  
Enrique Rudiño-Piñera ◽  
Alfonso Lira-Rocha ◽  
Alfredo Torres-Larios ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Aldehyde Dehydrogenase ◽  
Active Site ◽  
Cysteine Residue ◽  
Covalent Modification ◽  
Enzyme Inactivation ◽  
Betaine Aldehyde Dehydrogenase ◽  
Betaine Aldehyde ◽  
Oxidative Stresses ◽  
Covalent Adduct

PaBADH (Pseudomonas aeruginosa betaine aldehyde dehydrogenase) catalyses the irreversible NAD(P)+-dependent oxidation of betaine aldehyde to its corresponding acid, the osmoprotector glycine betaine. This reaction is involved in the catabolism of choline and in the response of this important pathogen to the osmotic and oxidative stresses prevalent in infection sites. The crystal structure of PaBADH in complex with NADPH showed a novel covalent adduct between the C2N of the pyridine ring and the sulfur atom of the catalytic cysteine residue, Cys286. This kind of adduct has not been reported previously either for a cysteine residue or for a low-molecular-mass thiol. The Michael addition of the cysteine thiolate in the ‘resting’ conformation to the double bond of the α,β-unsaturated nicotinamide is facilitated by the particular conformation of NADPH in the active site of PaBADH (also observed in the crystal structure of the Cys286Ala mutant) and by an ordered water molecule hydrogen bonded to the carboxamide group. Reversible formation of NAD(P)H–Cys286 adducts in solution causes reversible enzyme inactivation as well as the loss of Cys286 reactivity towards thiol-specific reagents. This novel covalent modification may provide a physiologically relevant regulatory mechanism of the irreversible PaBADH-catalysed reaction, preventing deleterious decreases in the intracellular NAD(P)+/NAD(P)H ratios.

Reversible, partial inactivation of plant betaine aldehyde dehydrogenase by betaine aldehyde: mechanism and possible physiological implications

2016 ◽  
Vol 473 (7) ◽  
pp. 873-885 ◽  
Author(s):  
Andrés Zárate-Romero ◽  
Darío S. Murillo-Melo ◽  
Carlos Mújica-Jiménez ◽  
Carmina Montiel ◽  
Rosario A. Muñoz-Clares
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Cysteine Residue ◽  
Betaine Aldehyde Dehydrogenase ◽  
Short Term ◽  
Betaine Aldehyde ◽  
Partial Inactivation ◽  
Reversible Formation

The activity of plant BADH enzymes may be down-regulated in the short term by a novel and physiologically relevant mechanism, consisting of the reversible formation of a thiohemiacetal between a conserved non-essential cysteine residue and the substrate betaine aldehyde.

Disulfiram irreversibly aggregates betaine aldehyde dehydrogenase—A potential target for antimicrobial agents against Pseudomonas aeruginosa

2006 ◽  
Vol 341 (2) ◽  
pp. 408-415 ◽  
Author(s):  
Roberto Velasco-García ◽  
Víctor J. Zaldívar-Machorro ◽  
Carlos Mújica-Jiménez ◽  
Lilian González-Segura ◽  
Rosario A. Muñoz-Clares
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Aldehyde Dehydrogenase ◽  
Antimicrobial Agents ◽  
Betaine Aldehyde Dehydrogenase ◽  
Betaine Aldehyde ◽  
Potential Target
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