A Conserved Histidine Residue of Escherichia Coli Outer‐Membrane Phospholipase A is Important for Activity

Abstract

Es:sr.herichia coli outer-membrane phospholipase A (OMPLA) is thought to be a tnember of the class of serine hydrolases, having a classical Asp-His-Ser catalytic triad [Horrevoets, A. J. G., Verheij, k1. M. & de Haas, G. H. (199'1) Eicr: .I. Biocherrr. 198, 247-2531. To idenlify the histidinc residue that is important for catalytic activity, the four histidine residues in E. c d i OMPLA that arc conserved in other enterobacterial OMPLA enzymes werc replaced by cysteine residues using PCR-directed, site-specific mutagenesis. The resulting mutant proteins were all well cxpressed and displayed heat modifiability, indicating that they were properly folded. Enzyme assays showed that only the Hisl42Cys mutant protein was lacking enzymatic activity. In addition, a His142Gly mutant protein appeared to he inactive. These result? show that His142 is important for the enzymatic activity of OMPLA.Keqrwords: outer-membrane protein; phospholipase A; active site; site-directed mutagenesis; Escherichiu coli.Outer-membrane phospholipase A (OMPLA) of Es:Jheric.hiti coli is a 30-kDa protein that is eiicodcd by the p1dA gene. It is widely disseminated among members of the Enterobacteriaceae family (Brok et al., 1994), but its physiological role is still unknown. It has been shown that R. coli OMPLA is required for efficient secretion of bacteriocins (Pugsley and Schwiutn, 1984; Luirink et al., 1986), but it is unlikely that this is the primary [unction of the protein. OMPLA activity must be well regulated since otherwise the enzyme would degrade the cell envelope. In normally growing cells, OMPLA appears to be dormant (A~idet et al., 1974). However, high OMPLA activity can be induced by damaging the membrane, e.g. by phage-induced lysis (Cronan and Wulff, 1960) or by temperalure shock (de Geus et al., 19x3). The 1~ldA genes of E. coli (Homtnii et al., 1984), Snlm!pplonell~i typhimurium, Klebsiellci pneurnoniuf,, and Proteus vulgnris (Rrok et al., 1094) have been cloned and sequenced, and they show high mutual sequence similarity.OMPLA has several enzymatic activities, i.e. those of phospholipases A, and A,, 1 -acyl-and 2-acyl-lysophospholipase, and diacylglycerol lipase, with (he phospholipase A, activity being six times higher thnn the phospholipase A, activity (Horrevoets et nl., 1989). It has bcen suggested (Horrevoets et al., 1991) that OMPLA belongs 10 thc class of serine hydrolases whose members catalyse cster hydrolysis via an ucyl cnzyme interniediate. However, in contrast to serine proteases (Kraut, 1977), OMPI>A is not inactivated by water-solublc serine hydrolase inhibitors like diisopropyltluorophosphate and phcnylmethylsulfotiyl fluoride. Only water-insoluble inhibitors, like n-hexadecylsulfonyl C~f~rrt..s~~o~~rluncr to H. M. Verheij,

A critical role for the histidine residues in the catalytic function of acyl‐CoA:cholesterol acyltransferase catalysis: Evidence for catalytic difference between ACAT1 and ACAT2

“…Histidine residues are frequently found in general acid-base catalysis [28,29], reaction intermediate stabilization [30][31][32] through hydrogen bonding of their NE2 nitrogen atom. Recently, the X-ray structure has been determined of OMPLA [33], an integral membrane enzyme, where the active center is located in the outer leaflet of the membrane and contains a histidine residue as part of the catalytic triad [34]. Another structural study of 3beta-hydroxysteroid dehydrogenase (3bHSD) isoenzymes demonstrated Fig.…”

Topology of the outer membrane phospholipase A of Salmonella typhimurium

“…Furthermore, PLST-L4E has no or extremely low specific activity. This is expected, since the insert is present close to residues His-142, Ser-144, and Ser-152, which have been shown to be important for enzymatic activity (8,9,24). Accessibility of the inserted epitopes.…”

Identification of FkpA as a Key Quality Control Factor for the Biogenesis of Outer Membrane Proteins under Heat Shock Conditions

“…All the electrophoresis was performed with 10% acrylamide gels. For the effective transfer and detection of the folded oligomers of OMPs that are wrapped by lipopolysaccharides, the gel was heated by steaming for 10 min before the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane for immunoblotting analysis (21,36).…”