title: Site-directed mutagenesis and homology modeling indicate an important role of cysteine 439 in the stability of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa.
abstract: Betaine aldehyde dehydrogenase (BADH) from the human pathogen Pseudomonas aeruginosa is a tetrameric enzyme that contains a catalytic Cys286 and three additional cysteine residues, Cys353, 377, and 439, per subunit. In the present study, we have investigated the role of the three non-essentials in enzyme activity and stability by homology modeling and site-directed mutagenesis. Cys353 and Cys377 are located at the protein surface with their sulfur atoms buried, while Cys439 is at the subunit interface between the monomers forming a dimeric pair. All three residues were individually mutated to alanine and Cys439 also to serine and valine. The five mutant proteins were expressed in Escherichia coli and purified to homogeneity. Their steady-state kinetics was not significantly affected, neither was their structure as indicated by circular dicroism spectropolarimetry, protein intrinsic fluorescence, and size-exclusion chromatography. However, stability was severely reduced in the Cys439 mutants particularly in C439S and C439V, which were inactive when expressed at 37 degrees C. They also exhibited higher sensitivity to thermal and chemical inactivation, and higher propensity to dissociation by dilution or exposure to low ionic strength than the wild-type enzyme. Size-exclusion chromatography indicates that substitution of Cys439 lead to unstable dimers or to stable dimeric conformations not compatible with a stable tetrameric structure. To the best of our knowledge, this is the first study of an aldehyde dehydrogenase revealing a residue at the dimer interface involved in holding the dimer, and consequently the tetramer, together.
authors.affiliation: Laboratorio de Osmorregulación, FES Iztacala, Av de los Barrios 1, Col. Los Reyes Iztacala, Tlalnepantla, Edo México 54090, Mexico.
authors.forename: Lilian
authors.initials: L
authors.lastname: González-Segura
authors.forename: Roberto
authors.initials: R
authors.lastname: Velasco-García
authors.forename: Enrique
authors.initials: E
authors.lastname: Rudiño-Piñera
authors.forename: Carlos
authors.initials: C
authors.lastname: Mújica-Jiménez
authors.forename: Rosario A
authors.initials: RA
authors.lastname: Muñoz-Clares
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D050728:Betaine-Aldehyde Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002942:Circular Dichroism; D003596:Cytosine; D005399:Fishes; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; D003596:Cytosine; D050728:Betaine-Aldehyde Dehydrogenase