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H C Wu - One of the best experts on this subject based on the ideXlab platform.

  • [49] Modification of bacterial lipoproteins
    Methods in Enzymology, 1995
    Co-Authors: Krishnan Sankaran, Sita D. Gupta, H C Wu
    Abstract:

    Publisher Summary This chapter summarizes current knowledge of the lipid modification enzymes in the pathway for the maturation of bacterial lipoproteins. Lipid modification of bacterial lipoproteins is catalyzed by three enzymes: diacylglyceryltransferase, Signal Peptidase II, and N -acyltransferase. Major phospholipids in the bacterial cell envelope serve as both the diacylglyceryl and acyl donors. Phosphatidylglycerol (PG) is the major diacylglyceryl donor for diacylglyceryltransferase, whereas N -acyltransferase does not exhibit a preference for any particular acyl donor. The ability of Tricine- sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) to separate the various intermediates in the biosynthesis of murein lipoprotein is exploited to develop assays for diacylglyceryl modification of prolipoprotein and for N -acylation of apolipoprotein. A simpler and quicker peptide-based assay for diacylglyceryltransferase is available and a similar one for N -acyltransferase is required for its purification. The availability of the genes for the enzymes should enable hyper expression and the development of simple purification protocols. The specificity toward PG in vivo is demonstrated in vitro by delipidating the inverted vesicles with aqueous acetone extraction followed by incubation of the delipidated enzyme preparations with individual phospholipid species.

  • Lipid modification of bacterial prolipoprotein. Transfer of diacylglyceryl moiety from phosphatidylglycerol.
    Journal of Biological Chemistry, 1994
    Co-Authors: Krishnan Sankaran, H C Wu
    Abstract:

    Abstract The peptide, MKATKLVLGAVILGSTLLAGCSSN, corresponding to the N-terminal 24 amino acids of Braun's prolipoprotein, was used to study the lipid modification of prolipoprotein in Escherichia coli by measuring the rate of incorporation of either [2-3H]glycerol or [9,10-3H]palmitate from the corresponding labeled phosphatidylglycerol into the peptide. Using E. coli strains containing varying levels of prolipoprotein diacylglyceryl modification activities due to mutations in or overexpression of the gene involved in diacylglyceryl modification (lgt), we have shown that the activities based on the peptide assay correlated well with the prolipoprotein-based assay. Further, we have followed the fate of the lipid substrate, phosphatidylglycerol, during the modification reaction and found that lipid modification of prolipoprotein involves the transfer of diacylglyceryl moiety from phosphatidylglycerol to the sulfhydryl group of the cysteine residue with the concomitant formation of sn-glycerol 1-phosphate. This mechanism is contrary to the previously proposed two-step mechanism of an initial glyceryl transferase followed by O-acyl transfer (Chattopadhyay, P.K., and Wu, H.C. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5318-5322). Accordingly, the enzyme that catalyzes this activity has been named phosphatidylglycerol-prolipoprotein diacylglyceryl transferase. The revised pathway for the lipoprotein biogenesis in bacteria consists of three successive reactions catalyzed by prolipoprotein diacylglyceryl transferase, Signal Peptidase II, and apolipoprotein N-acyltransferase.

  • Processing of lipid-modified prolipoprotein requires energy and sec gene products in vivo.
    Journal of Bacteriology, 1993
    Co-Authors: N. Kosic, M. Sugai, H C Wu
    Abstract:

    Abstract The kinetics of processing of glyceride-modified prolipoprotein that accumulated in globomycin-treated Escherichia coli has been found to be affected by sec mutations, i.e., secA, secE, secY, secD, and secF, and by metabolic poisons which affect proton motive force (PMF). The effect of sec mutations on processing of glyceride-modified prolipoprotein in vivo was not due to a secondary effect on PMF. Neither a secF mutation nor metabolic poisons affected the processing of previously accumulated proOmpA protein in vivo, suggesting that the requirements for functional sec gene products and PMF are specific to the processing of lipoprotein precursors by Signal Peptidase II.

  • membrane topology of escherichia coli prolipoprotein Signal Peptidase Signal Peptidase II
    Journal of Biological Chemistry, 1991
    Co-Authors: F J Munoa, K W Miller, R Beers, M Graham, H C Wu
    Abstract:

    Abstract The lsp gene of Escherichia coli encodes the inner membrane enzyme, Signal Peptidase II (SPase II). SPase II is comprised of 164 amino acid residues and contains four hydrophobic domains. A series of lsp-phoA and lsp-lacZ gene fusions have been constructed in vitro to determine the topology of SPase II. The fusion junction for each of these gene fusions was determined by DNA sequencing. The lengths of the SPase II fragment in the fusions varied from 12 to 159 amino acid residues. Strains containing SPase II-PhoA fusions to the two predicted periplasmic loops exhibited higher levels of alkaline phosphatase activity than fusions to the predicted cytoplasmic domains. In contrast, SPase II-LacZ fusions at the cytoplasmic and the periplasmic domains of SPase II showed high and low levels of beta-galactosidase activity, respectively, a result opposite to those shown by SPase II-PhoA fusions located at precisely the same amino acid of SPase II. Taken together, these results strongly support the predicted model for SPase II topology, i.e. this enzyme spans the cytoplasmic membrane four times with both the amino and the carboxyl termini facing the cytoplasm.

  • nucleotide sequence of the pseudomonas fluorescens Signal Peptidase II gene lsp and flanking genes
    Journal of Bacteriology, 1990
    Co-Authors: L Isaki, R Beers, H C Wu
    Abstract:

    The lsp gene encoding prolipoprotein Signal Peptidase (Signal Peptidase II) is organized into an operon consisting of ileS and three open reading frames, designated genes x, orf149, and orf316 in both Escherichia coli and Enterobacter aerogenes. A plasmid, pBROC128, containing a 5.8-kb fragment of Pseudomonas fluorescens DNA was found to confer pseudomonic acid resistance on E. coli host cells and to contain the structural gene of ileS from P. fluorescens. In addition, E. coli strains carrying pBROC128 exhibited increased globomycin resistance. This indicated that the P. fluorescens lsp gene was present on the plasmid. The nucleotide sequences of the P. fluorescens lsp gene and of its flanking regions were determined. Comparison of the nucleotide sequences of the lsp genes in E. coli and P. fluorescens revealed two highly conserved domains in this enzyme. Furthermore, the five genes which constitute an operon in E. coli and Enterobacter aerogenes were found in P. fluorescens in the same order as in the first two species.

R Beers - One of the best experts on this subject based on the ideXlab platform.

  • membrane topology of escherichia coli prolipoprotein Signal Peptidase Signal Peptidase II
    Journal of Biological Chemistry, 1991
    Co-Authors: F J Munoa, K W Miller, R Beers, M Graham, H C Wu
    Abstract:

    Abstract The lsp gene of Escherichia coli encodes the inner membrane enzyme, Signal Peptidase II (SPase II). SPase II is comprised of 164 amino acid residues and contains four hydrophobic domains. A series of lsp-phoA and lsp-lacZ gene fusions have been constructed in vitro to determine the topology of SPase II. The fusion junction for each of these gene fusions was determined by DNA sequencing. The lengths of the SPase II fragment in the fusions varied from 12 to 159 amino acid residues. Strains containing SPase II-PhoA fusions to the two predicted periplasmic loops exhibited higher levels of alkaline phosphatase activity than fusions to the predicted cytoplasmic domains. In contrast, SPase II-LacZ fusions at the cytoplasmic and the periplasmic domains of SPase II showed high and low levels of beta-galactosidase activity, respectively, a result opposite to those shown by SPase II-PhoA fusions located at precisely the same amino acid of SPase II. Taken together, these results strongly support the predicted model for SPase II topology, i.e. this enzyme spans the cytoplasmic membrane four times with both the amino and the carboxyl termini facing the cytoplasm.

  • nucleotide sequence of the pseudomonas fluorescens Signal Peptidase II gene lsp and flanking genes
    Journal of Bacteriology, 1990
    Co-Authors: L Isaki, R Beers, H C Wu
    Abstract:

    The lsp gene encoding prolipoprotein Signal Peptidase (Signal Peptidase II) is organized into an operon consisting of ileS and three open reading frames, designated genes x, orf149, and orf316 in both Escherichia coli and Enterobacter aerogenes. A plasmid, pBROC128, containing a 5.8-kb fragment of Pseudomonas fluorescens DNA was found to confer pseudomonic acid resistance on E. coli host cells and to contain the structural gene of ileS from P. fluorescens. In addition, E. coli strains carrying pBROC128 exhibited increased globomycin resistance. This indicated that the P. fluorescens lsp gene was present on the plasmid. The nucleotide sequences of the P. fluorescens lsp gene and of its flanking regions were determined. Comparison of the nucleotide sequences of the lsp genes in E. coli and P. fluorescens revealed two highly conserved domains in this enzyme. Furthermore, the five genes which constitute an operon in E. coli and Enterobacter aerogenes were found in P. fluorescens in the same order as in the first two species.

  • cloning and nucleotide sequence of the enterobacter aerogenes Signal Peptidase II lsp gene
    Journal of Bacteriology, 1990
    Co-Authors: L Isaki, R Beers, M Kawakami, H C Wu
    Abstract:

    In Escherichia coli, prolipoprotein Signal Peptidase is encoded by the lsp gene, which is organized into an operon consisting of ileS, lsp, and three open reading frames, designated genes x, orf-149, and orf-316. The Enterobacter aerogenes lsp gene was cloned and expressed in E. coli. The nucleotide sequence of the Enterobacter aerogenes lsp gene and a part of its flanking sequences were determined. A high degree of homology was found between the E. coli ileS-lsp operon and the corresponding genes in Enterobacter aerogenes. Furthermore, the same five genes which constitute an operon in E. coli were found in Enterobacter aerogenes in the same order. Images

Henry C Wu - One of the best experts on this subject based on the ideXlab platform.

  • nucleotide sequence of the staphylococcus aureus Signal Peptidase II lsp gene
    FEBS Letters, 1992
    Co-Authors: Xiaojiong Zhao, Henry C Wu
    Abstract:

    The lsp gene encoding prolipoprotein Signal Peptidase (Signal Peptidase II) of Staphylococcus aureus was cloned by screening a genomic library for plasmid clones capable of complementing a conditionally lethal lsp allele of Escherichia coli, E. coli cells carrying one of five overlapping clones exhibited increased resistance to globomycin. The nucleotide sequence of the S. aureus lsp gene was determined. The deduced amino acid sequence of the Signal Peptidase II of S. aureus suggests that this enzyme has a hydropathy profile very similar to those of E. coli, Enterobacter aerogenes and Pseudomonas fluorescens. Comparison of the primary structures of this enzyme from these four distinct bacterial species reveals three highly conserved domains in proteins which have a low degree of overall sequence homology. Unlike the lsp genes from the Gram-negative bacteria, the lsp gene in S. aureus is not flanked by x-ileS and orf149-orf316 as found in E. coli, Ent. aerogenes, and P. fluorescens.

  • Lipoproteins in bacteria
    Journal of Bioenergetics and Biomembranes, 1990
    Co-Authors: Shigeru Hayashi, Henry C Wu
    Abstract:

    Covalent modification of membrane proteins with lipids appears to be ubiquitous in all living cells. The major outer membrane (Braun's) lipoprotein ofE. coli, the prototype of bacterial lipoproteins, is first synthesized as a precursor protein. Analysis of Signal sequences of 26 distinct lipoprotein precursors has revealed a consensus sequence of lipoprotein modification/processing site of Leu-(Ala, Ser)-(Gly, Ala)-Cys at − 3 to + 1 positions which would represent the cleavage region of about three-fourth of all lipoprotein Signal sequences in bacteria. Unmodified prolipoprotein with the putative consensus sequence undergoes sequential modification and processing reactions catalyzed by glyceryl transferase, O-acyl transferase(s), prolipoprotein Signal Peptidase (Signal Peptidase II), and N-acyl transferase to form mature lipoprotein. Like all exported proteins, the export of lipoprotein requires functional SecA, SecY, and SecD proteins. Thus all precursor proteins are exported through a common pathway accessible to both Signal Peptidase I and Signal Peptidase II. The rapidly increasing list of lipid-modified proteins in both prokaryotic as well as eukaryotic cells indicates that lipoproteins comprise a diverse group of structurally and functionally distinct proteins. They share a common structural feature which is derived from a common biosynthetic pathway.

Jan Maarten Van Dijl - One of the best experts on this subject based on the ideXlab platform.

  • Active lipoprotein precursors in the Gram-positive eubacterium Lactococcus lactis
    Journal of Biological Chemistry, 2003
    Co-Authors: Roelke Venema, Harold Tjalsma, Jan Maarten Van Dijl, Anne De Jong, Kees Leenhouts, Girbe Buist, Gerard Venema
    Abstract:

    Lipid-modified proteins play important roles at the interface between eubacterial cells and their environment. The importance of lipoprotein processing by Signal Peptidase II (SPase II) is underscored by the fact that this enzyme is essential for viability of the Gram-negative eubacterium Escherichia coli. In contrast, SPase II is not essential for growth and viability of the Gram-positive eubacterium Bacillus subtilis. This could be due to alternative amino-terminal lipoprotein processing, which was shown previously to occur in SPase II mutants of B. subtilis. Alternatively, uncleaved lipoprotein precursors might be functional. To explore further the importance of lipoprotein processing in Gram-positive eubacteria, an SPase II mutant strain of Lactococcus lactis was constructed. Although some of the 39 (predicted) lactococcal lipoproteins, such as PrtM and OppA, are essential for growth in milk, the growth of SPase II mutant L. lactis cells in this medium was not affected. Furthermore, the activity of the strictly PrtM-dependent extracellular protease PrtP, which is required for casein degradation, was not impaired in the absence of SPase II. Importantly, no alternative processing of pre-PrtM and pre-OppA was observed in cells lacking SPase II. Taken together, these findings show for the first time that authentic lipoprotein precursors retain biological activity.

  • the role of lipoprotein processing by Signal Peptidase II in the gram positive eubacterium bacillus subtilis Signal Peptidase II is required for the efficient secretion of alpha amylase a non lipoprotein
    Journal of Biological Chemistry, 1999
    Co-Authors: Harold Tjalsma, Vesa Kontinen, Zoltan Pragai, Matti Sarvas, Hongyan Wu, Sierd Bron, Rob Meima, Gerard Venema, Jan Maarten Van Dijl
    Abstract:

    Computer-assisted analyses indicate that Bacillus subtilis contains approximately 300 genes for exported proteins with an amino-terminal Signal peptide. About 114 of these are lipoproteins, which are retained in the cytoplasmic membrane. We have investigated the importance of lipoprotein processing by Signal Peptidase II (SPase II) for cellular homeostasis, using cells lacking SPase II. The results show that lipoprotein processing is important for cell viability at low and high temperatures, suggesting that lipoproteins are essential for growth under these conditions. Although certain lipoproteins are required for the development of genetic competence, sporulation, and germination, these developmental processes were not affected in the absence of SPase II. Cells lacking SPase II accumulated lipid-modified precursor and mature-like forms of PrsA, a folding catalyst for secreted proteins. These forms of PrsA seem to have a reduced activity, as the secretion of a-amylase was strongly impaired. Unexpectedly, type I Signal Peptidases, which process secretory preproteins, were not involved in alternative amino-terminal processing of pre-PrsA in the absence of SPase II. In conclusion, processing of lipoproteins by SPase II in B. subtilis is not strictly required for lipoprotein function, which is surprising as lipoproteins and type II SPases seem to be conserved in all eubacteria.

  • The Signal Peptidase II (Isp) gene of Bacillus subtilis.
    Microbiology (Reading England), 1997
    Co-Authors: Zoltan Pragai, Harold Tjalsma, Jan Maarten Van Dijl, Gerard Venema, Albert Bolhuis, Sierd Bron
    Abstract:

    The gene encoding the type II Signal Peptidase (SPase II) of Bacillus subtilis was isolated by screening a genomic DNA library of this bacterium for the ability to increase the levels of globomycin resistance in Escherichia coli, and to complement the growth deficiency at the non-permissive temperature of E. coli strain Y815 carrying a temperature-sensitive mutation in its Isp gene for SPase II. The deduced amino acid sequence of the B. subtilis SPase II showed significant similarity with those of other known SPase II enzymes. Activity of the B. subtilis SPase II was demonstrated by a pulse-labelling experiment in E. coli. In B. subtilis, the Isp gene is flanked by the isoleucyl-tRNA synthetase (ileS) gene and the pyrimidine biosynthetic (pyr) gene cluster, which is known to map at 139 degrees of the chromosome. In the Gram-positive bacteria studied thus far, Isp appears to be the first gene in an operon. The promoter-distal gene ("orf4') of this operon specifies a hypothetical protein in bacteria and yeast.

Hiroshi Kogen - One of the best experts on this subject based on the ideXlab platform.

  • Structure-activity relationships of globomycin analogues as antibiotics.
    Bioorganic & Medicinal Chemistry, 2004
    Co-Authors: Toshihiro Kiho, Mizuka Nakayama, Kayo Yasuda, Shunichi Miyakoshi, Masatoshi Inukai, Hiroshi Kogen
    Abstract:

    Abstract Globomycin (1a), a Signal Peptidase II inhibitor, and its derivatives show potent antibacterial activity against Gram-negative bacteria. The synthesis and antimicrobial activity of novel globomycin analogues are reported. The hydroxyl group in the l -Ser residue was essential for the antimicrobial activity and the length of the alkyl side chain greatly influenced the activity. In addition, derivatives that had a modified cyclic core exhibited weak activity. One of the analogues showed a wider antimicrobial spectrum, effective against not only Gram-negative but also Gram-positive bacteria.

  • Synthesis and antimicrobial activity of novel globomycin analogues
    Bioorganic & Medicinal Chemistry Letters, 2003
    Co-Authors: Toshihiro Kiho, Mizuka Nakayama, Kayo Yasuda, Shunichi Miyakoshi, Masatoshi Inukai, Hiroshi Kogen
    Abstract:

    Globomycin, a Signal Peptidase II inhibitor, and its derivatives show potent antibacterial activity against Gram-negative bacteria. The synthesis and antimicrobial activity of novel globomycin analogues are reported. One of the analogues showed a more potent activity against Gram-negative bacteria than globomycin and also exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA).

  • total synthesis and nmr conformational study of Signal Peptidase II inhibitors globomycin and sf 1902 a5
    Tetrahedron, 2003
    Co-Authors: Toshihiro Kiho, Mizuka Nakayama, Hiroshi Kogen
    Abstract:

    Abstract A stereoselective total synthesis of an antibiotic, globomycin ( 1a ), and its congener, SF-1902 A5 ( 1b ), was achieved. Two convergent macrocyclization routes via macrolactamization or macrolactonization to form 1a are described. A conformational study by means of NMR spectroscopy was performed in several solvents. The 1H NMR spectrum of 1a indicated that the amide proton of only the l -allo-Thr residue was involved in the hydrogen bonding. The structure in solution phase was different from the X-ray structure.

  • conformational analysis of globomycin with a Signal Peptidase II inhibitory activity using molecular dynamics simulation
    Drug Design and Discovery, 2003
    Co-Authors: Toshihiro Kiho, Yoriko Iwata, Hiroshi Kogen, Shuichi Miyamoto
    Abstract:

    Globomycin (1), a 19-membered cyclic depsipeptide, exhibited an antibiotic activity against gram-negative bacteria by inhibiting Signal Peptidase II in the cytoplasmic membrane. Although only one conformation of 1 was observed for the crystal structure, it was revealed by 1H NMR spectroscopic analysis that 1 exists as a mixture of two rotational isomers in solution (CDCl3 and CD3OD). A conformational analysis of 1 was, therefore, performed by high-temperature molecular dynamics simulation in combination with 1H NMR analysis to elucidate the conformations in solution. The relative ratio of the major and minor isomers present, which differs depending on the solvent, was then derived from their relative energy differences obtained in the conformational analysis. The difference in the relative ratios corresponded with that calculated from the 1H NMR analysis. Finally, the predicted conformations in solution were compared with that of the X-ray crystal structure to find local and global differences that charac...