共查询到20条相似文献,搜索用时 15 毫秒
1.
Daniela Mavrici Daniil M. Prigozhin Tom Alber 《Protein science : a publication of the Protein Society》2014,23(4):481-487
Resuscitation promoting factor (Rpf) proteins, which hydrolyze the sugar chains in cell‐wall peptidoglycan (PG), play key roles in prokaryotic cell elongation, division, and escape from dormancy to vegetative growth. Like other bacteria, Mycobacterium tuberculosis (Mtb) expresses multiple Rpfs, none of which is individually essential. This redundancy has left unclear the distinct functions of the different Rpfs. To explore the distinguishing characteristics of the five Mtb Rpfs, we determined the crystal structure of the RpfE catalytic domain. The protein adopts the characteristic Rpf fold, but the catalytic cleft is narrower compared to Mtb RpfB. Also in contrast to RpfB, in which the substrate‐binding surfaces are negatively charged, the corresponding RpfE catalytic pocket and predicted peptide‐binding sites are more positively charged at neutral pH. The complete reversal of the electrostatic potential of the substrate‐binding site suggests that the different Rpfs function optimally at different pHs or most efficiently hydrolyze different micro‐domains of PG. These studies provide insights into the molecular determinants of the evolution of functional specialization in Rpfs. 相似文献
2.
van Straaten KE Dijkstra BW Vollmer W Thunnissen AM 《Journal of molecular biology》2005,352(5):1068-1080
Lytic transglycosylases are bacterial enzymes involved in the maintenance and growth of the bacterial cell-wall peptidoglycan. They cleave the beta-(1,4)-glycosidic bonds in peptidoglycan forming non-reducing 1,6-anhydromuropeptides. The crystal structure of the lytic transglycosylase MltA from Escherichia coli without a membrane anchor was solved at 2.0A resolution. The enzyme has a fold completely different from those of the other known lytic transglycosylases. It contains two domains, the largest of which has a double-psi beta-barrel fold, similar to that of endoglucanase V from Humicola insolens. The smaller domain also has a beta-barrel fold topology, which is weakly related to that of the RNA-binding domain of ribosomal proteins L25 and TL5. A large groove separates the two domains, which can accommodate a glycan strand, as shown by molecular modelling. Several conserved residues, one of which is in a position equivalent to that of the catalytic acid of the H.insolens endoglucanase, flank this putative substrate-binding groove. Mutation of this residue, Asp308, abolished all activity of the enzyme, supporting the direct participation of this residue in catalysis. 相似文献
3.
Bacterial peptidoglycan amidases are a large and diverse group of enzymes. During the last few years, genomic sequence information has accumulated to an extent such that lists of proven or predicted peptidoglycan amidases can now be expected to be fairly complete. Moreover, representative crystal structures for most groups of phylogenetically related peptidoglycan amidases have been solved. Here, sequence and structural information is combined with published biochemical findings to demonstrate that (a) peptidoglycan amidases have evolved for almost every bond that occurs in peptidoglycan, (b) there are enzymes that share the fold, yet cleave different bonds and (c) there are enzymes that have entirely different folds and must have evolved independently, and yet cleave the same peptide bond. It is shown that despite these complications, some rules can be deduced from the available biochemical and structural information that can be useful to predict the specificity of hypothetical peptidoglycan hydrolases, for which only sequence information is available. 相似文献
4.
Abstract The endopeptidase(s) of ether-treated cells of Neisseria gonorrhoeae cleaved O -acetylated peptidoglycan dimers about 3 times as rapidly as non- O -acetylated dimers. This was true whether the enzyme came from a highly O -acetylated strain or from one with low O -acetylation. The penicillin-resistant fraction of the endopeptidase activity also showed the same substrate selectivity. 相似文献
5.
Proteins of the Rpf (resuscitation promoting factor) family are peptidoglycan hydrolases 总被引:18,自引:0,他引:18
Telkov MV Demina GR Voloshin SA Salina EG Dudik TV Stekhanova TN Mukamolova GV Kazaryan KA Goncharenko AV Young M Kaprelyants AS 《Biochemistry. Biokhimii?a》2006,71(4):414-422
The secreted Micrococcus luteus protein, Rpf, is required for successful resuscitation of dormant "non-culturable" M. luteus cells and for growth stimulation in poor media. The biochemical mechanism of Rpf action remained unknown. Theoretical predictions of Rpf domain architecture and organization, together with a recent NMR analysis of the protein structure, indicate that the conserved Rpf domain has a lysozyme-like fold. In the present study, we found that both the secreted native protein and the recombinant protein lyse crude preparations of M. luteus cell walls. They also hydrolyze 4-methylumbelliferyl-beta-D-N,N',N'-triacetylchitotrioside, a synthetic substrate for peptidoglycan muramidases, with optimum activity at pH 6. The Rpf protein also has weak proteolytic activity against N-CBZ-Gly-Gly-Arg-beta-naphthylamide, a substrate for trypsin-like enzymes. Rpf activity towards 4-methylumbelliferyl-beta-D-N,N',N'-triacetylchitotrioside was reduced when the glutamate residue at position 54, invariant for all Rpf family proteins and presumably involved in catalysis, was altered. The same amino acid substitution resulted in impaired resuscitation activity of Rpf. The data indicate that Rpf is a peptidoglycan-hydrolyzing enzyme, and strongly suggest that this specific activity is responsible for its growth promotion and resuscitation activity. A possible mechanism of Rpf-mediated resuscitation is discussed. 相似文献
6.
Penicillin-binding proteins (PBPs) have been scrutinized for over 40 years. Recent structural information on PBPs together with the ongoing long-term biochemical experimental investigations, and results from more recent techniques such as protein localization by green fluorescent protein-fusion immunofluorescence or double-hybrid assay, have brought our understanding of the last stages of the peptidoglycan biosynthesis to an outstanding level that allows a broad outlook on the properties of these enzymes. Details are emerging regarding the interaction between the peptidoglycan-synthesizing PBPs and the peptidoglycan, their mesh net-like product that surrounds and protects bacteria. This review focuses on the detailed structure of PBPs and their implication in peptidoglycan synthesis, maturation and recycling. An overview of the content in PBPs of some bacteria is provided with an emphasis on comparing the biochemical properties of homologous PBPs (orthologues) belonging to different bacteria. 相似文献
7.
Bacterial epoxide hydrolases of opposite enantiopreference 总被引:1,自引:0,他引:1
Wolfram Krenn Ingrid Osprian Wolfgang Kroutil Gerhart Braunegg Kurt Faber 《Biotechnology letters》1999,21(8):687-690
Epoxide hydrolases of matching opposite enantiopreference were found among various Actinomyces spp. While (S)-2,2-disubstituted oxiranes were hydrolyzed by Rhodococcus and Nocardia spp., several strains of methylotrophic bacteria, such as Mycoplana rubra and Methylobacterium spp., exhibited a preference for the (R)-enantiomers. Thus, the stereochemical course of the reaction can be controlled by a simple choice of the appropriate enzyme source. 相似文献
8.
Ingrid Bourgeois Emilie Camiade Raja Biswas Pascal Courtin Laure Gibert Friedrich Götz Marie-Pierre Chapot-Chartier Jean-Louis Pons & Martine Pestel-Caron 《FEMS microbiology letters》2009,290(1):105-113
The nucleotide sequence of atlL , a gene encoding a putative Staphylococcus lugdunensis peptidoglycan hydrolase, was determined using degenerate consensus PCR and genome walking. This 3837-bp gene encodes a protein, AtlL, that appears as a putative bifunctional autolysin with a 29-amino acid putative signal peptide and two enzymatic putative centres ( N -acetylmuramoyl- l -alanine amidase and N -acetylglucosaminidase) interconnected with three imperfect repeated sequences displaying glycine–tryptophan motifs. In order to determine whether both lytic domains were functional, and verify their exact enzymatic activities, gene fragments harbouring both putative domains, AM ( N -acetylmuramoyl- l -alanine amidase enzymatic centre plus two repeated sequences) and GL ( N -acetylglucosaminidase enzymatic centre plus one repeated sequence), were isolated, subcloned, and expressed in Escherichia coli . Purified recombinant AM and GL protein truncations exhibited cell wall lytic activity in zymograms performed with cell walls of Micrococcus lysodeikticus, Bacillus subtilis , and S. lugdunensis. AtlL is expressed during the whole growth, with an overexpression in the early-exponential stage. Liquid chromatography-mass spectrometry analysis of muropeptides generated by digestion of B. subtilis cell walls demonstrated the hydrolytic bond specificities and confirmed both of the acetyl domains' activities as predicted by sequence homology data. AtlL is the first autolysin described in S. lugdunensis , with a bifunctional enzymatic activity involved in peptidoglycan hydrolysis. 相似文献
9.
A system of intracellular peptidoglycan hydrolases of Xanthomonas campestris XL-1 comprises about 10 enzymes of different localization and substrate specificity. Seven enzymes (A1-A7) are localized in cytosol, one enzyme (A8) in periplasm, and two enzymes (A9, A10) were found in the fraction of cell walls and membranes. While the culture is entering the logarithmic growth stage from the stationary stage, a change occurs in the activity of the cytosolic enzymes: A1 significantly increases, and A5 and A6 decrease. The spectrum of cytosolic enzymes also depends on the growth medium composition. The enzyme A7 present in cells secreting extracellular enzymes (medium 5/5) was not found in non-secreting cells (LB medium). Unlike extracellular enzymes, intracellular peptidoglycan hydrolases are primarily acidic proteins. The data indicate that the system of intracellular peptidoglycan hydrolases of X. campestris is under complex and strict regulation. 相似文献
10.
Paradis-Bleau C Cloutier I Lemieux L Sanschagrin F Laroche J Auger M Garnier A Levesque RC 《FEMS microbiology letters》2007,266(2):201-209
The gp144 endolysin gene from the Pseudomonas aeruginosa phage phiKZ was cloned and studies of gp144 expression into Escherichia coli showed host cell lysis. The gp144 protein was purified directly from the culture supernatant and from the bacterial cell pellet and showed in vitro antibacterial lytic activity against P. aeruginosa bacteria and degraded purified peptidoglycan of Gram-negative bacteria. MS analysis identified the gp144 peptidoglycan cleavage site and confirmed a lytic transglycosylase enzyme. Studies of gp144 expression in the presence of sodium azide (NaN(3)), an inhibitor of the protein export machinery, and into an E. coli MM52 secA(ts) mutant at permissive and restrictive temperatures showed that gp144 was secreted independently of the Sec system. The solution conformation of purified gp144 analyzed by circular dichroism spectroscopy was 61% in alpha-helical content, and showed a 72% decrease when interacting with dimyristoylphosphatidylglycerol (DMPG), one of the major components of bacterial membranes and less than 10% with dimyristoylphosphatidylcholine (DMPC) found in eukaryotic membranes. Membrane vesicles of DMPG anionic lipids containing calcein indicated that gp144 caused a rapid release of fluorescent calcein when interacting with synthetic membranes. These results indicated that gp144 from phiKZ is a lytic transglycosylase capable of interacting with and disorganizing bacterial membranes and has potential as an antipseudomonal in phage therapy. 相似文献
11.
Extracellular proteolytic activation of bacteriolytic peptidoglycan hydrolases of Staphylococcus simulans biovar staphylolyticus 总被引:3,自引:0,他引:3
Veronica C. Neumann Harry E. Heath Paul A. LeBlanc Gary L. Sloan 《FEMS microbiology letters》1993,110(2):205-211
Abstract Staphylococcus simulans biovar staphylolyticus secreted two bacteriolytic peptidoglycan hydrolases as proproteins that were activated as they were processed by an extracellular sulphydryl protease. This processing resulted in the production of multiple molecular-mass forms of each enzyme. Cells from early exponential phase cultures were susceptible to lysis by the mature forms of each of the peptidoglycan hydrolases whereas stationary phase cells were resistant. Thus secretion of these bacteriolytic enzymes during early exponential growth as precursors that are activated later by the protease would provide time for the cells to become resistant. 相似文献
12.
Abstract We have isolated some mould strains that can grow under acid conditions with poly(3-hydroxybutyrate) (PHB) as sole carbon source, and secrete PHB hydrolases active at pH values at least down to 3. An improved assay method for such enzymes using a pH stat has been developed, and used to determine the dependence of reaction rate on enzyme and polymer concentrations. The implications of these kinetic properties of the PHB hydrolase for its mode of action are discussed. 相似文献
13.
Andrius Jasilionis Magdalena Plotka Lei Wang Sebastian Dorawa Joanna Lange Hildegard Watzlawick Tom van den Bergh Bas Vroling Josef Altenbuchner AnnaKarina Kaczorowska Ehmke Pohl Tadeusz Kaczorowski Eva Nordberg Karlsson Stefanie FreitagPohl 《Protein science : a publication of the Protein Society》2023,32(3)
Bacteriophages encode a wide variety of cell wall disrupting enzymes that aid the viral escape in the final stages of infection. These lytic enzymes have accumulated notable interest due to their potential as novel antibacterials for infection treatment caused by multiple‐drug resistant bacteria. Here, the detailed functional and structural characterization of Thermus parvatiensis prophage peptidoglycan lytic amidase AmiP, a globular Amidase_3 type lytic enzyme adapted to high temperatures is presented. The sequence and structure comparison with homologous lytic amidases reveals the key adaptation traits that ensure the activity and stability of AmiP at high temperatures. The crystal structure determined at a resolution of 1.8 Å displays a compact α/β‐fold with multiple secondary structure elements omitted or shortened compared with protein structures of similar proteins. The functional characterization of AmiP demonstrates high efficiency of catalytic activity and broad substrate specificity toward thermophilic and mesophilic bacteria strains containing Orn‐type or DAP‐type peptidoglycan. The here presented AmiP constitutes the most thermoactive and ultrathermostable Amidase_3 type lytic enzyme biochemically characterized with a temperature optimum at 85°C. The extraordinary high melting temperature T m 102.6°C confirms fold stability up to approximately 100°C. Furthermore, AmiP is shown to be more active over the alkaline pH range with pH optimum at pH 8.5 and tolerates NaCl up to 300 mM with the activity optimum at 25 mM NaCl. This set of beneficial characteristics suggests that AmiP can be further exploited in biotechnology. 相似文献
14.
塑料自20世纪首次合成以来给人类生活带来了极大的便利。然而,塑料稳定的高分子结构导致了塑料废弃物的持续堆积,对生态环境和人类健康均造成严重威胁。聚对苯二甲酸乙二醇酯[poly(ethylene terephthalate),PET]是产量最高的一种聚酯类塑料,近年来PET水解酶的相关研究展现出生物酶法对塑料进行降解、回收的巨大潜力,也为塑料生物降解机制研究建立了参考范例。本文综述了不同微生物来源的PET水解酶及其PET降解能力,阐述了最具代表性的PET水解酶—IsPETase降解PET的催化机理,并总结了近年来通过酶工程改造而获得的高效降解酶,为未来的PET降解机制研究、PET高效降解酶的进一步挖掘和改造提供参考。 相似文献
15.
【目的】旨在研究光滑鳖甲Anatolica polita肽聚糖识别蛋白(peptidoglycan recognition proteins, PGRPs)基因ApPGRP表达模式及其对下游免疫相关基因的表达调控,以及重组蛋白ApPGRP与细菌的结合能力。【方法】构建原核表达载体pET-28a-ApPGRP,转化大肠杆菌Escherichia coli BL21(DE3),诱导表达并纯化重组蛋白His-ApPGRP,分别测定其与金黄色葡萄球菌Staphylococcus aureus和肽聚糖(peptidoglycan, PGN)的结合能力。金黄色葡萄球菌S.aureus刺激后,利用qRT-PCR检测光滑鳖甲6龄幼虫体内ApPGRP、抗菌肽基因ApAttacin2和ApAttacin1、防御素基因ApDefensin、丝氨酸蛋白酶基因ApSP和丝氨酸蛋白酶抑制剂基因ApSerpin等免疫相关基因的表达水平;采用RNAi技术沉默光滑鳖甲6龄幼虫体内ApPGRP基因的表达,并利用qRT-PCR分别检测RNAi处理以及RNAi后再用S.aureus刺激时幼虫体内上述基因的表达水平变化。【结果】通过原核表达获得了重组蛋白His-ApPGRP,其具有结合金黄色葡萄球菌和肽聚糖的能力。注射金黄色葡萄球菌后,检测的光滑鳖甲6龄幼虫免疫相关基因(ApSP除外)的表达都显著上调;RNAi沉默光滑鳖甲6龄幼虫ApPGRP后,其他免疫相关基因表达量均显著降低,其响应金黄色葡萄球菌刺激后的表达量也显著低于对照组。【结论】这些结果表明,ApPGRP在光滑鳖甲免疫防御中起着识别外源微生物,激活信号通路并调控抗菌肽表达的作用。 相似文献
16.
Bacillus anthracis produces metabolically inactive spores. Germination of these spores requires germination‐specific lytic enzymes (GSLEs) that degrade the unique cortex peptidoglycan to permit resumption of metabolic activity and outgrowth. We report the first crystal structure of the catalytic domain of a GSLE, SleB. The structure revealed a transglycosylase fold with unique active site topology and permitted identification of the catalytic glutamate residue. Moreover, the structure provided insights into the molecular basis for the specificity of the enzyme for muramic‐δ‐lactam‐containing cortex peptidoglycan. The protein also contains a metal‐binding site that is positioned directly at the entrance of the substrate‐binding cleft. Proteins 2012;. © 2012 Wiley Periodicals, Inc. 相似文献
17.
MltA is a lytic transglycosylase of Gram-negative bacteria that cleaves the beta-1,4 glycosidic linkages between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in peptidoglycan. We have determined the crystal structures of MltA from Neisseria gonorrhoeae and Escherichia coli (NgMltA and EcMltA), which have only 21.5% sequence identity. Both proteins have two main domains separated by a deep groove. Domain 1 shows structural similarity with the so-called double-psi barrel family of proteins. Comparison of the two structures reveals substantial differences in the relative positions of domains 1 and 2 such that the active site groove in NgMltA is much wider and appears more able to accommodate peptidoglycan substrate than EcMltA, suggesting that domain closure occurs after substrate binding. Docking of a peptidoglycan molecule into the structure of NgMltA reveals a number of conserved residues that are likely involved in substrate binding, including a potential binding pocket for the peptidyl moieties. This structure supports the assignment of Asp405 as the acid catalyst responsible for cleavage of the glycosidic bond. In EcMltA, the equivalent residue is Asp328, which has been identified previously. The structures also suggest a catalytic role for Asp393 (Asp317 in EcMltA) in activating the C6 hydroxyl group during formation of the 1,6-anhydro linkage. Finally, in comparison to EcMltA, NgMltA contains a unique third domain that is an insertion within domain 2. The domain is beta in structure and may mediate protein-protein interactions that are specific to peptidoglycan metabolism in N.gonorrhoeae. 相似文献
18.
Crystal structure of the Drosophila peptidoglycan recognition protein (PGRP)-SA at 1.56 A resolution
Peptidoglycan recognition proteins (PGRPs) form a recently discovered protein family, which is conserved from insect to mammals and is implicated in the innate immune system by interacting with/or degrading microbial peptidoglycans (PGNs). Drosophila PGRP-SA is a member of this family of pattern recognition receptors and is involved in insect Toll activation. We report here the crystal structure of PGRP-SA at 1.56 A resolution, which represents the first example of a "recognition" PGRP. Comparison with the catalytic Drosophila PGRP-LB reveals an overall structure conservation with an L-shaped hydrophilic groove that is likely the PGN carbohydrate core binding site, but further suggests some possible functional homology between recognition and catalytic PGRPs. Consistent with sequence analysis, PGRP-SA does not contain the canonical zinc-binding residues found in catalytic PGRPs. However, substitution of the zinc-binding cysteine residue by serine, along with an altered coordinating histidine residue, assembles a constellation of residues that resembles a modified catalytic triad. The serine/histidine juxtaposition to a threonine residue and a carbonyl oxygen atom, along with conservation of the catalytic water molecule found in PGRP-LB, tantalizingly suggests some hydrolytic function for this member of receptor PGRPs. 相似文献
19.
【目的】肽聚糖识别蛋白(peptidoglycan recognition proteins,PGRPs)是昆虫免疫系统中一类重要的模式识别蛋白。本研究旨在阐明经苏云金芽孢杆菌Bacillus thuringiensis侵染后,小菜蛾Plutella xylostella PGRP-SA基因(命名为Px PGRP-SA)在体内的表达模式和对抗菌肽基因的表达调控。【方法】本研究利用实时荧光定量PCR(qRT-PCR)技术分析B.thuringiensis侵染小菜蛾幼虫后Px PGRP-SA的转录模式,通过RNAi技术结合抗血清封闭实验检测Px PGRP-SA对小菜蛾抗菌肽基因的表达调控作用。【结果】qRT-PCR检测表明,小菜蛾4龄幼虫在注射具有活性的B.thuringiensis 6 h后,Px PGRP-SA在脂肪体和血细胞中表达量迅速上升,其中脂肪体中的表达量在注射24 h后达到高峰,而在血细胞中的表达量在18 h后达到高峰。RNAi沉默小菜蛾4龄幼虫Px PGRP-SA的转录后,可显著降低小菜蛾脂肪体中cecropin,moricin-2,lysozyme和defensin 4个抗菌肽基因及Dorsal和Sptzle基因的mRNA转录水平;注射anti-Px PGRP-SA封闭小菜蛾体内Px PGRP-SA的活性后,也可降低小菜蛾脂肪体中4个抗菌肽基因的mRNA转录水平;Px PGRP-SA转录沉默后,同时导致添食B.thuringiensis的小菜蛾幼虫的存活率明显降低。【结论】Px PGRP-SA参与了小菜蛾体内抗菌肽cecropin,moricin-2,lysozyme和defensin基因的表达调控,并在免疫防御B.thuringiensis的侵染过程中起了重要的作用。 相似文献
20.
Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind peptidoglycans (PGNs) of bacterial cell walls. These molecules, which are highly conserved from insects to mammals, contribute to host defense against infections by both Gram-positive and Gram-negative bacteria. Here, we present the crystal structure of human PGRP-S at 1.70A resolution. The overall structure of PGRP-S, which participates in intracellular killing of Gram-positive bacteria, is similar to that of other PGRPs, including Drosophila PGRP-LB and PGRP-SA and human PGRP-Ialpha. However, comparison with these PGRPs reveals important differences in both the PGN-binding site and a groove formed by the PGRP-specific segment on the opposite face of the molecule. This groove, which may constitute a binding site for effector or signaling proteins, is less hydrophobic and deeper in PGRP-S than in PGRP-IalphaC, whose PGRP-specific segments vary considerably in amino acid sequence. By docking a PGN ligand into the PGN-binding cleft of PGRP-S based on the known structure of a PGRP-Ialpha-PGN complex, we identified potential PGN-binding residues in PGRP-S. Differences in PGN-contacting residues and interactions suggest that, although PGRPs may engage PGNs in a similar mode, structural differences exist that likely regulate the affinity and fine specificity of PGN recognition. 相似文献