PubMed · 6371900
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T Yoshimoto, D Tsuru. 1984. [Proline specific endopeptidase].. https://pubmed.ncbi.nlm.nih.gov/6371900/
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The Burkholderia cepacia epidemic strain marker (BCESM) is a useful epidemiological marker for virulent B. cenocepacia strains that infect patients with cystic fibrosis. However, there was no evidence that the original marker, identified by random amplified polymorphic DNA fingerprinting, contributed to pathogenicity. Here we demonstrate that the BCESM is part of a novel genomic island encoding genes linked to both virulence and metabolism. The BCESM was present on a 31.7-kb low-GC-content island that encoded 35 predicted coding sequences (CDSs): an N-acyl homoserine lactone (AHL) synthase gene (cciI) and corresponding transcriptional regulator (cciR), representing the first time cell signaling genes have been found on a genomic island; fatty acid biosynthesis genes; an IS66 family transposase; transcriptional regulator CDSs; amino acid metabolism genes; and a group of hypothetical genes. Mutagenesis of the AHL synthase, amidase (amiI), and porin (opcI) genes on the island was carried out. Testing of the isogenic mutants in a rat model of chronic lung infection demonstrated that the amidase played a role in persistence, while the AHL synthase and porin were both involved in virulence. The island, designated the B. cenocepacia island (cci), is the first genomic island to be defined in the B. cepacia complex and its discovery validates the original epidemiological correlation of the BCESM with virulent CF strains. The features of the cci, which overlap both pathogenicity and metabolism, expand the concept of bacterial pathogenicity islands and illustrate the diversity of accessory functions that can be acquired by lateral gene transfer in bacteria.
Polymyxin acylase isolated from Pseudomonas sp. M-6-3 was used as an N-myristoyl cleaving enzyme in order to determine a part of the N-terminal amino acid sequence of N-myristoyl proteins. The enzyme hydrolyzed a number of N-myristoyl oligopeptides at various hydrolysis rates but not N-myristoyl proteins. The oncogenic protein (N-myristoyl-pp60c-src) was isolated from human colon adenocarcinoma cell line COLO 320DM by two-dimensional polyacrylamide gel electrophoresis. The protein was digested with trypsin and the resultant tryptic N-myristoyl tetrapeptide (N-myristoyl-Gly-Ser-Asn-Lys) was purified by HPLC and the structure was determined both by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MASS) and by a gas-phase protein sequencer before or after treatment with the polymyxin acylase. The results suggest that the N-myristoyl peptide sequence derived from N-myristoyl proteins was clearly determined by the combined use of MALDI TOF MASS and the N-myristoyl cleaving enzyme.
Crystallographic analysis and site-directed mutagenesis have been used to identify the catalytic and oligosaccharide recognition residues of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F (PNGase F), an amidohydrolase that removes intact asparagine-linked oligosaccharide chains from glycoproteins and glycopeptides. Mutagenesis has shown that three acidic residues, Asp-60, Glu-206, and Glu-118, that are located in a cleft at the interface between the two domains of the protein are essential for activity. The D60N mutant has no detectable activity, while E206Q and E118Q have less than 0.01 and 0.1% of the wild-type activity, respectively. Crystallographic analysis, at 2.0-A resolution, of the complex of the wild-type enzyme with the product, N,N'-diacetylchitobiose, shows that Asp-60 is in direct contact with the substrate at the cleavage site, while Glu-206 makes contact through a bridging water molecule. This indicates that Asp-60 is the primary catalytic residue, while Glu-206 probably is important for stabilization of reaction intermediates. Glu-118 forms a hydrogen bond with O6 of the second N-acetylglucosamine residue of the substrate and the low activity of the E118Q mutant results from its reduced ability to bind the oligosaccharide. This analysis also suggests that the mechanism of action of PNGase F differs from those of L-asparaginase and glycosylasparaginase, which involve a threonine residue as the nucleophile.