title: A conserved histidine residue of Escherichia coli outer-membrane phospholipase A is important for activity.
abstract: Escherichia coli outer-membrane phospholipase A (OMPLA) is thought to be a member of the class of serine hydrolases, having a classical Asp-His-Ser catalytic triad [Horrevoets, A. J. G., Verheij, H. M. & de Haas, G. H. (1991) Eur. J. Biochem. 198, 247-253]. To identify the histidine residue that is important for catalytic activity, the four histidine residues in E. coli OMPLA that are conserved in other enterobacterial OMPLA enzymes were replaced by cysteine residues using PCR-directed, site-specific mutagenesis. The resulting mutant proteins were all well expressed and displayed heat modifiability, indicating that they were properly folded. Enzyme assays showed that only the His142Cys mutant protein was lacking enzymatic activity. In addition, a His142Gly mutant protein appeared to be inactive. These results show that His142 is important for the enzymatic activity of OMPLA.
authors.affiliation: Institute of Biomembranes, Utrecht University, The Netherlands.
authors.forename: R G
authors.initials: RG
authors.lastname: Brok
authors.forename: N
authors.initials: N
authors.lastname: Dekker
authors.forename: N
authors.initials: N
authors.lastname: Gerrits
authors.forename: H M
authors.initials: HM
authors.lastname: Verheij
authors.forename: J
authors.initials: J
authors.lastname: Tommassen
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D017124:Conserved Sequence; D017931:DNA Primers; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D006639:Histidine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D013696:Temperature
chemical_list: D017931:DNA Primers; D006639:Histidine; D010741:Phospholipases A