The pid4 gene of Escherichia coli encodes an outer membrane phospholipase A. A strain carrying the most commonly used mutant pkld allele appeared to express a correctly assembled PldA protein in the outer membrane. Nucleotide sequence analysis revealed that the only difference between the wild type and the mutant is the replacement of the serine residue in position 152 by phenylalanine. Since mutants that lack thepld4 gene were normally viable under laboratory conditions and had no apparent phenotype except for the lack of outer membrane phospholipase activity, the exact role of the enzyme remains unknown. Nevertheless, the enzyme seems to be important for the bacteria, since Western blotting (immunoblotting) and enzyme assays showed that it is widely spread among species of the family Enterobacteriaceae. To characterize the PldA protein further, the pid4 genes of Salmonella typhimurium, Klebsiella pneumoniae, and Proteus vulgaris were cloned and sequenced. The cloned genes were expressed in E. coli, and their gene products were enzymatically active. Comparison of the predicted PldA primary structures with that of E. coli PldA revealed a high degree of homology, with 79%o of the amino acid residues being identical in all four proteins. Implications of the sequence comparison for the structure and the structure-function relationship of PldA protein are discussed.Most bacterial outer membrane proteins are involved in the transport of nutrients across this membrane by forming pores or receptors. In addition, these membranes contain a few enzymes, e.g., the detergent-resistant outer membrane phospholipase A or PldA protein of Escherichia coli. Several activities reside in this enzyme, i.e., those of phospholipases Al and A2 and of 1-acyl and 2-acyl lysophospholipase and lipase, with the phospholipase A1 activity being six times greater than the phospholipase A2 activity (24). The PIdA protein is encoded by the pldA gene, the nucleotide sequence of which has been determined (23). This gene codes for a 30-kDa mature protein of 269 amino acid residues preceded by a signal sequence of 20 amino acid residues. The three-dimensional structure of the enzyme is unknown. Like other outer membrane proteins, PldA protein lacks hydrophobic sequences long enough to span the lipid bilayer. Therefore, its structure might be comparable with those of the porins which have recently been determined (10,54,55). In these outer membrane proteins, the polypeptide chain traverses the outer membrane repeatedly as antiparallel 3-strands. A comparison of the outer membrane protein PhoE among three species of the family Enterobacteriaceae has revealed that during evolution some parts of the polypeptide have undergone more extensive divergence than others (49). For PhoE, these variable regions correspond to cell surface-exposed segments. A similar finding was reported for the OmpA protein (7). Thus, sequence comparisons can be helpful in predicting the topology of outer membrane proteins.The exact function of the PldA protein is unknown. The pro...

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The structure of the central repetitive domain of high molecular weight (HMW) wheat gluten proteins was characterized in solution and in the dry state using HMW proteins Bx6 and Bx7 and a subcloned, bacterially expressed part of the repetitive domain of HMW Dx5. Model studies of the HMW consensus peptides PGQGQQ and GYYPTSPQQ formed the basis for the data analysis (van Dijk AA et al., 1997, Protein Sci 6:637-648). In solution, the repetitive domain contained a continuous nonoverlapping series of both type I and type I1 p-turns at positions predicted from the model studies; type I1 p-turns occurred at QPGQ and QQGY sequences and type I p-turns at YPTS and SPQQ. The subcloned part of the HMW Dx5 repetitive domain sometimes migrated as two bands on SDS-PAGE; we present evidence that this may be caused by a single amino acid insertion that disturbs the regular structure of p-turns. The type I p-turns are lost when the protein is dried on a solid surface, probably by conversion to type I1 p-turns. The homogeneous type I1 p-turn distribution is compatible with the formation of a P-spiral structure, which provides the protein with elastic properties. The p-turns and thus the P-spiral are stabilized by hydrogen bonds within and between turns. Reformation of this hydrogen bonding network after, e.g., mechanical disruption may be important for the elastic properties of gluten proteins.Keywords: elastic properties; repetitive proteins; structural characterization; wheat gluten proteins The central repetitive domain of high molecular weight proteins forms 60-80% of their amino acid sequence and is built from the consensus peptides PGQGQQ, GYYPTS(P/L)QQ, and GQQ Shewry et al., 1994). Its structural organization may consist of a series of p-turns that organize in a @spiral structure (Tatham et al., 1984;Shewry et al., 1994); this idea is supported by scanning tunneling microscopy images of HMW Dx5 (Miles et al., 1991). The proposed &spiral structure for HMW proteins is similar to that of elastin, where it forms the basis for the molecule's elastic properties (Uny, 1993). The structural analogy between HMW proteins and elastin suggests that the HMW proteins might have elastic properties as well; however, no Reprint requests to: G.T. Robillard, Department of Biochemistry and Biophysical Chemistry and the Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands; e-mail: G.T.Robillard@chem.rg.nl.'Present address: Gist Brocades N.V. Postbus 1, 2600 MA Delft, The Netherlands.Abbreviurions: FITR, fourier transform infrared; HMW, high molecular weight; IPTG, isopropyl-P-D-thiogalactopyranoside. detailed structural characterization of the HMW repetitive domain has been performed. In the companion paper (van Dijk et al., 1997), we described the structure of the consensus sequences of the repetitive domain using cyclic and linear peptides. We showed that they are highly structured and contain a mixture of type I and I1 p-turns that are stabilized by mu...

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