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

• Published in: World journal of microbiology & biotechnology 2013-06, Vol.29 (6), p.991-999
• Main Authors: Campos-Garcia, Jesus, Diaz-Perez, Cesar, Diaz-Perez, Alma Laura
• Format: Article
• Language: English
• Subjects: Amino acids; Amino Acids - genetics; Amino Acids - metabolism; Analysis; Applied Microbiology; Bacteria; Biochemistry; Biomedical and Life Sciences; Biotechnology; Catalysis; Catalytic Domain; Cloning; Cluster Analysis; Computational Biology; Conserved Sequence; DNA Mutational Analysis; E coli; Environmental Engineering/Biotechnology; Enzymatic activity; Enzyme Stability; Enzymes; Fatty acids; Genes; Isocitrate Lyase - chemistry; Isocitrate Lyase - genetics; Isocitrate Lyase - metabolism; Life Sciences; Microbiology; Models, Molecular; Mutagenesis; Mutagenesis, Site-Directed; Mutation; Original Paper; Phylogenetics; Phylogeny; Plasmids; Protein Conformation; Protein expression; Protein Stability; Proteins; Pseudomonas aeruginosa; Pseudomonas aeruginosa - enzymology; Pseudomonas aeruginosa - genetics; Residues; Sequence Homology; Studies; Thermal properties
• ISSN: 0959-3993; 1573-0972
• DOI: 10.1007/s11274-013-1258-8

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 Q IE N Q V S DE K Q CGH QD. 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.