title: Residues Asn214, Gln211, Glu219 and Gln221 contained in the subfamily 3 catalytic signature of the isocitrate lyase from Pseudomonas aeruginosa are involved in its catalytic and thermal properties.
abstract: Isocitrate lyase, encoded by the aceA gene, plays an important role in the ability of Pseudomonas aeruginosa to grow on fatty acids, acetate, acyclic terpenes, and amino acids. Phylogenetic analysis indicated that the ICL superfamily is divided in two families: the ICL family, which includes five subfamilies, and the 2-methylisocitrate lyase (MICL) family. ICL from P. aeruginosa (ICL-Pa) was identified in a different ICL node (subfamily 3) than other Pseudomonas ICL enzymes (grouped in subfamily 1). Analysis also showed that psychrophilic bacteria are mainly grouped in ICL subfamily 3, whose ICL proteins contain the highly conserved catalytic pattern QIENQVSDEKQCGHQD. We performed site-directed mutagenesis, enzymatic activity, and structure modeling of conserved residues in mutated ICLs by using ICL-Pa as a model. Our results indicated that the N214 residue is essential for catalytic function, while mutating the Q211, E219, and Q221 residues impairs its catalytic and thermostability properties. Our findings suggest that conserved residues in the subfamily 3 signature of ICL-Pa play important roles in catalysis and thermostability and are likely associated with the catalytic loop structural conformation.
authors: Laboratorio de Biotecnologia, Instituto de Investigaciones Quimico-Biologicas, Universidad Michoacana de San Nicolas de Hidalgo, 58030 Morelia, Michoacan, Mexico. jcgarcia@umich.mx
authors: Jesus
authors: J
authors: Campos-Garcia
authors: Cesar
authors: C
authors: Diaz-Perez
authors: Alma Laura
authors: AL
authors: Diaz-Perez
mesh_terms: D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D016000:Cluster Analysis; D019295:Computational Biology; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D007522:Isocitrate Lyase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D011487:Protein Conformation; D055550:Protein Stability; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics*; D017385:Sequence Homology
chemical_list: D000596:Amino Acids; D007522:Isocitrate Lyase