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

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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Characterization of an Autoreduction Pathway for the [Fe4S4]3+Cluster of MutantChromatium vinosumHigh-Potential Iron Proteins. Site-Directed Mutagenesis Studies To Probe the Role of Phenylalanine 66 in Defining the Stability of the [Fe4S4] Center Provide Evidence for Oxidative Degradation via a [Fe3S4] Cluster†

Biochemistry ◽

10.1021/bi961658l ◽

1996 ◽

Vol 35 (46) ◽

pp. 14544-14552 ◽

Cited By ~ 13

Author(s):

Shumin Bian ◽

C. F. Hemann ◽

Russ Hille ◽

J. A. Cowan

Keyword(s):

Oxidative Degradation ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Iron Proteins ◽

The Stability

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Role of the Highly Conserved Histidine Residues in Rat Liver Mitochondrial Aldehyde Dehydrogenase as Studied by Site-Directed Mutagenesis

Archives of Biochemistry and Biophysics ◽

10.1006/abbi.1993.1447 ◽

1993 ◽

Vol 305 (2) ◽

pp. 460-466 ◽

Cited By ~ 6

Author(s):

C.F. Zheng ◽

H. Weiner

Keyword(s):

Rat Liver ◽

Aldehyde Dehydrogenase ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Histidine Residues

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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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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

Biochimie ◽

10.1016/j.biochi.2010.09.022 ◽

2011 ◽

Vol 93 (2) ◽

pp. 286-295 ◽

Cited By ~ 16

Author(s):

Víctor J. Zaldívar-Machorro ◽

Manuel López-Ortiz ◽

Patricia Demare ◽

Ignacio Regla ◽

Rosario A. Muñoz-Clares

Keyword(s):

Pseudomonas Aeruginosa ◽

Aldehyde Dehydrogenase ◽

Bacterial Growth ◽

Betaine Aldehyde Dehydrogenase ◽

Betaine Aldehyde

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Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01703-15 ◽

2016 ◽

Vol 60 (5) ◽

pp. 3123-3126 ◽

Cited By ~ 5

Author(s):

Carlo Bottoni ◽

Mariagrazia Perilli ◽

Francesca Marcoccia ◽

Alessandra Piccirilli ◽

Cristina Pellegrini ◽

...

Keyword(s):

Catalytic Activity ◽

Catalytic Efficiency ◽

Kinetic Studies ◽

Zinc Ion ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

The Stability

ABSTRACTSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-β-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

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