The Crystal Structure of A Ternary Complex of Betaine Aldehyde Dehydrogenase from Pseudomonas aeruginosa Provides New Insight into the Reaction Mechanism and Shows A Novel Binding Mode of the 2′-Phosphate of NADP+ and A Novel Cation Binding Site

Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 Å resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of theZoogloea ramigerabiosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2–Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.

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

Biochemical Journal ◽

10.1042/bj3520675 ◽

2000 ◽

Vol 352 (3) ◽

pp. 675-683 ◽

Cited By ~ 21

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.

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

Biochimie ◽

10.1016/j.biochi.2005.06.007 ◽

2005 ◽

Vol 87 (12) ◽

pp. 1056-1064 ◽

Cited By ~ 13

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

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

Archives of Microbiology ◽

10.1007/s00203-005-0054-8 ◽

2005 ◽

Vol 185 (1) ◽

pp. 14-22 ◽

Cited By ~ 27

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

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Crystal structure of holoenolase refined at 1.9 .ANG. resolution: trigonal-bipyramidal geometry of the cation binding site

Journal of the American Chemical Society ◽

10.1021/ja00204a030 ◽

1989 ◽

Vol 111 (22) ◽

pp. 8511-8513 ◽

Cited By ~ 31

Author(s):

Lukasz Lebioda ◽

Boguslaw Stec

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Cation Binding ◽

Trigonal Bipyramidal Geometry ◽

Trigonal Bipyramidal ◽

Cation Binding Site

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

Biochemical Journal ◽

10.1042/bj20110376 ◽

2011 ◽

Vol 439 (3) ◽

pp. 443-455 ◽

Cited By ~ 14

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.

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