scholarly journals Characterization of the sporulation-related γ-d-glutamyl-(l)meso-diaminopimelic-acid-hydrolysing peptidase I of Bacillus sphaericus NCTC 9602 as a member of the metallo(zinc) carboxypeptidase A family. Modular design of the protein

1993 ◽  
Vol 292 (2) ◽  
pp. 563-570 ◽  
Author(s):  
M L Hourdou ◽  
M Guinand ◽  
M J Vacheron ◽  
G Michel ◽  
L Denoroy ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Modular Design ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Protein A ◽  
Bacillus Sphaericus ◽  
Diaminopimelic Acid ◽  
Carboxypeptidase A ◽  
Terminal Repeats

The sporulation-related gamma-D-glutamyl-(L)meso-diaminopimelic-acid-hydrolysing peptidase I of Bacillus sphaericus NCTC 9602 has been analysed by proton-induced X-ray emission. It contains 1 equivalent Zn2+ per mol of protein. As derived from gene cloning and sequencing, the B. sphaericus Zn peptidase I is a two-module protein. A 100-amino-acid-residue N-terminal domain consisting of two tandem segments of similar sequences, is fused to a 296-amino-acid-residue C-terminal catalytic domain. The catalytic domain belongs to the Zn carboxypeptidase A family, the closest match being observed with the Streptomyces griseus carboxypeptidase [Narahashi (1990) J. Biochem. 107, 879-886] and with the family prototype, bovine carboxypeptidase A. The catalytic domain of the B. sphaericus peptidase I possesses, distributed along the amino-acid sequence, peptide segments, a triad His162-Glu165-His307 and a dyad Tyr347-Glu366 that are equivalent to secondary structures, the zinc-binding triad His69-Glu72-His196 and the catalytic dyad Tyr248-Glu270 of bovine carboxypeptidase A respectively. The N-terminal repeats of the B. sphaericus peptidase I have similarity with the C-terminal repeats of the Enterococcus hirae muramidase 2, the Streptococcus (now Enterococcus) faecalis autolysin and the Bacillus phi PZA and phi 29 lysozymes, to which a role in the recognition of a particular moiety of the bacterial cell envelope has been tentatively assigned. Detergents enhance considerably the specific activity of the B. sphaericus peptidase I.

Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase

2001 ◽  
Vol 47 (12) ◽  
pp. 1088-1094 ◽  
Author(s):  
Yew-Loom Chen ◽  
Tsung-Yin Tang ◽  
Kuo-Joan Cheng
Keyword(s):  
Amino Acid ◽  
Directed Evolution ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Synergistic Effects ◽  
High Specific Activity ◽  
Site Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Neocallimastix Patriciarum

The catalytic domain of a xylanase from the anaerobic fungus Neocallimastix patriciarum was made more alkalophilic through directed evolution using error-prone PCR. Transformants expressing the alkalophilic variant xylanases produced larger clear zones when overlaid with high pH, xylan-containing agar. Eight amino acid substitutions were identified in six selected mutant xylanases. Whereas the wild-type xylanase exhibited no activity at pH 8.5, the relative and specific activities of the six mutants were higher at pH 8.5 than at pH 6.0. Seven of the eight amino acid substitutions were assembled in one enzyme (xyn-CDBFV) by site-directed mutagenesis. Some or all of the seven mutations exerted positive and possibly synergistic effects on the alkalophilicity of the enzyme. The resulting composite mutant xylanase retained a greater proportion of its activity than did the wild type at pH above 7.0, maintaining 25% of its activity at pH 9.0, and its retention of activity at acid pH was no lower than that of the wild type. The composite xylanase (xyn-CDBFV) had a relatively high specific activity of 10 128 µmol glucose·min–1·(mg protein)–1 at pH 6.0. It was more thermostable at 60°C and alkaline tolerant at pH 10.0 than the wild-type xylanase. These properties suggest that the composite mutant xylanase is a promising and suitable candidate for paper pulp bio-bleaching.Key words: xylanase, Neocallimastix patriciarum, alkalophilicity, random mutagenesis, directed evolution.

scholarly journals The 8-amino-7-oxopelargonate synthase from Bacillus sphaericus. Purification and preliminary characterization of the cloned enzyme overproduced in Escherichia coli

1992 ◽  
Vol 283 (2) ◽  
pp. 327-331 ◽  
Author(s):  
O Ploux ◽  
A Marquet
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Absorption Band ◽  
Specific Activity ◽  
Bacillus Sphaericus ◽  
Complete Agreement ◽  
Amino Acid Residues ◽  
Characteristic Absorption Band ◽  
Preliminary Characterization

The 8-amino-7-oxopelargonate synthase [6-carboxyhexanoyl-CoA:L-alanine carboxyhexanoyltransferase (decarboxylating); EC 2.3.1.47] from Bacillus sphaericus involved in biotin biosynthesis was purified from an Escherichia coli overproducing strain. The purification afforded an electrophoretically homogeneous enzyme with a specific activity of 0.67 unit/mg. The purified enzyme is a monomer of 41 kDa. N-Terminal sequencing of the first 14 amino acid residues showed complete agreement with the predicted sequence from the bioF gene. The pure enzyme showed the characteristic absorption band (425 nm) of pyridoxal 5′-phosphate-dependent enzymes. Furthermore, the holoenzyme was resolved during an affinity step yielding the inactive apoenzyme, which recovered activity and the 425 nm-absorption band on dialysis against pyridoxal 5′-phosphate. Km values for L-alanine and pimeloyl-CoA were respectively 3 mM and 1 microM.

scholarly journals Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Qingping Xu ◽  
Dominique Mengin-Lecreulx ◽  
Xueqian W. Liu ◽  
Delphine Patin ◽  
Carol L. Farr ◽  
...  
Keyword(s):  
Cell Wall ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Catalytic Domain ◽  
Substrate Binding ◽  
Diaminopimelic Acid ◽  
Single Mutation ◽  
Molecular Architecture ◽  
Substrate Specificities ◽  
Cell Wall Hydrolases

ABSTRACTBacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.

scholarly journals Preparative isolation of the two forms of pig pancreatic pro-(carboxypeptidase A) and their monomeric carboxypeptidases A

1985 ◽  
Vol 229 (3) ◽  
pp. 605-609 ◽  
Author(s):  
M Vilanova ◽  
J Vendrell ◽  
M T López ◽  
C M Cuchillo ◽  
F X Avilés
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
High Specific Activity ◽  
High Yield ◽  
Binary Complex ◽  
Acid Hydrolase ◽  
Carboxypeptidase A ◽  
Preparative Isolation

A method is reported for the preparative isolation of the two forms of pro-(carboxypeptidase A) from pig pancreas: the monomer and the binary complex with pro-(proteinase E). This method, which is mainly based on chromatography on DEAE-Sepharose at pH 5.7, allows these proenzymes to be prepared more quickly and in safer conditions than with other reported methods. Undegraded and homogeneous carboxypeptidase A1 and A2 species (peptidyl-L-amino acid hydrolase, EC 3.4.17.1), in monomeric forms with high specific activity, are also obtained in high yield by controlled trypsin activation of either of the pro-(carboxypeptidases A) followed by chromatography on DEAE-Sepharose at pH 5.8 under dissociating conditions (7 M-urea).

scholarly journals Identification of MpaA, an Amidase in Escherichia coli That Hydrolyzes the γ-d-Glutamyl-meso-Diaminopimelate Bond in Murein Peptides

2003 ◽  
Vol 185 (2) ◽  
pp. 679-682 ◽  
Author(s):  
Tsuyoshi Uehara ◽  
James T. Park
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Double Mutant ◽  
Biosynthetic Pathway ◽  
Bacillus Sphaericus ◽  
Cell Extract ◽  
Diaminopimelic Acid ◽  
Multicopy Plasmid ◽  
Amino Acid Sequence Homology ◽  
Hydrolysis Of

ABSTRACT MpaA amidase was identified in Escherichia coli by its amino acid sequence homology with the ENP1 endopeptidase from Bacillus sphaericus. The enzymatic activity of MpaA, i.e., hydrolysis of the γ-d-glutamyl-diaminopimelic acid bond in the murein tripeptide l-alanyl-γ-d-glutamyl-meso-diaminopimelic acid, was demonstrated in the cell extract of a strain expressing mpaA from a multicopy plasmid. An mpaA mpl (murein peptide ligase) double mutant accumulated large amounts of murein tripeptide in its cytoplasm, consistent with the premise that MpaA degrades the tripeptide if its recycling via the peptidoglycan biosynthetic pathway is blocked.

scholarly journals Active site labelling of inositol 1,4,5-trisphosphate 3-kinase A by phenylglyoxal

1995 ◽  
Vol 310 (1) ◽  
pp. 109-115 ◽  
Author(s):  
D Communi ◽  
R Lecocq ◽  
V Vanweyenberg ◽  
C Erneux
Keyword(s):  
Amino Acid ◽  
Rat Brain ◽  
Catalytic Domain ◽  
Protein A ◽  
Peptide Sequence ◽  
Amino Acid Derivative ◽  
Atp Binding Site ◽  
Inositol 1,4,5 Trisphosphate ◽  
Rate Kinetics ◽  
Kinase A

Chemical modification by phenylglyoxal, an arginine-specific reagent, of both native and recombinant rat brain inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase A was accompanied by irreversible inhibition of enzyme activity. This effect was prevented in the presence of the substrate ATP but not Ins(1,4,5)P3. The modification reaction obeyed pseudo-first-order rate kinetics. Complete inhibition of activity corresponded to incorporation of 1.2 mol of phenylglyoxal per mol of protein. A single [14C]phenylglyoxal-modified peptide was isolated following alpha-chymotrypsin digestion of the radiolabelled Ins(1,4,5)P3 3-kinase and reverse-phase HPLC. ATP prevented the incorporation of radioactivity to this peptide. The peptide sequence (i.e. QWREGISSSTTL) corresponded to amino acids 315 to 326 of rat brain Ins(1,4,5)P3 3-kinase A. An estimate of the radioactivity of the different phenylthiohydantoin amino acid derivative showed the modified amino acid to be Arg-317. The data directly identify a reactive arginine residue as part of the ATP-binding site. Arg-317 is located within a sequence segment which is conserved among the catalytic domain of Ins(1,4,5)P3 3-kinase isoenzymes A and B in human and rat species.

scholarly journals Sakacin G, a New Type of Antilisterial Bacteriocin

2002 ◽  
Vol 68 (12) ◽  
pp. 6416-6420 ◽  
Author(s):  
L. Simon ◽  
C. Fremaux ◽  
Y. Cenatiempo ◽  
J. M. Berjeaud
Keyword(s):  
Antimicrobial Activity ◽  
Amino Acid ◽  
Amino Acid Residue ◽  
Specific Activity ◽  
Disulfide Bridges ◽  
Structural Genes ◽  
Lactobacillus Sake ◽  
Sequence Homologies ◽  
New Type

ABSTRACT Sakacin G is a 37-amino-acid-residue-long class IIa bacteriocin produced by Lactobacillus sake 2512, which is encoded by the duplicated structural genes skgA1 and skgA2. Sakacin G appears to be unique and seems to be an intermediate between pediocin-like bacteriocins, according to its double-disulfide bridges required for antimicrobial activity, and mesentericin-like bacteriocins in terms of sequence homologies, inhibition spectrum, and specific activity.

FRACTIONATION AND ANALYSIS OF MONKEY PITUITARY GLANDS FOR POSTERIOR LOBE HORMONES

1962 ◽  
Vol 40 (2) ◽  
pp. 283-289 ◽  
Author(s):  
Darrell N. Ward ◽  
Earl F. Walborg ◽  
Harry S. Lipscomb ◽  
Roger Guillemin
Keyword(s):  
Amino Acid ◽  
Current Distribution ◽  
Arginine Vasopressin ◽  
Specific Activity ◽  
Small Scale ◽  
Posterior Lobe ◽  
Pharmacological Activities ◽  
Pituitary Glands ◽  
Counter Current ◽  
Counter Current Distribution

ABSTRACT Fractionation of monkey pituitary glands gave an oxytocin fraction in low yield which showed a counter-current distribution coefficient equivalent to that obtained with oxytocin from other species. Fractionation and chromatography of monkey vasopressin on carboxymethyl cellulose gave arginine-vasopressin of 60% purity, based on amino acid analysis and specific activity. Counter-current distribution on a small scale gave arginine-vasopressin of 89% purity. Reports by others that monkey pituitary glands contain arginine-vasopressin, based on pharmacological activities, are substantiated by the chemical data presented here.

Sign in / Sign up

Export Citation Format

Share Document