scholarly journals Sheep mast-cell proteinases-1 and -3: cDNA cloning, primary structure and molecular modelling of the enzymes and further studies on substrate specificity

1998 ◽  
Vol 333 (3) ◽  
pp. 801-809 ◽  
Author(s):  
Sybil M. McALEESE ◽  
Alan D. PEMBERTON ◽  
Mary E. McGRATH ◽  
John F. HUNTLEY ◽  
Hugh R. P. MILLER
Keyword(s):  
Mast Cell ◽  
Amino Acid ◽  
Salt Concentration ◽  
Catalytic Domain ◽  
Serine Proteinase ◽  
Binding Pocket ◽  
Cathepsin G ◽  
Amino Acid Residues ◽  
Human Thrombin ◽  
Amino Acid Signal Peptide

Sheep mast-cell proteinase-1 (sMCP-1) is a serine proteinase expressed predominantly by mucosal mast cells, with specificity for cleavage C-terminal to basic and hydrophobic amino acid residues. A cDNA encoding sMCP-1 has been cloned using reverse transcriptase (RT)-PCR. It appears to be translated as a pre-proenzyme with a 17-amino-acid signal peptide, a basic 2-amino-acid propeptide and a 226-amino-acid catalytic domain. A second cDNA, encoding a serine proteinase 90% identical with sMCP-1, was also cloned and named sMCP-3. Molecular models were constructed for both enzymes using coordinates for the refined X-ray structures of human cathepsin G, chymase and rat mast-cell proteinase-2. The model for sMCP-1 suggests that the acidic Asp-226 side chain extends into the substrate-binding pocket, hydrogen-bonding with Ser-190 on the opposite side and bisecting the pocket. The location of an acidic moiety in this position would favour interaction with basic substrate residues and binding of aromatic residues is rationalized by interaction of the positively charged equatorial plane with Asp-226. The balance between chymotryptic and tryptic activities of sMCP-1 was found to be sensitive to salt concentration, with increasing univalent cation concentration favouring chymotryptic activity relative to the tryptic. Using a peptide substrate representing residues 36–59 of the human thrombin receptor, increasing salt concentration favoured cleavage at Phe-43 rather than at Arg-41.

scholarly journals Isolation and amino-acid sequence of two inhibitors of serine proteinases, members of the squash inhibitor family, from Echinocystis lobata seeds.

1996 ◽  
Vol 43 (3) ◽  
pp. 507-513 ◽  
Author(s):  
D Stachowiak ◽  
A Polanowski ◽  
G Bieniarz ◽  
T Wilusz
Keyword(s):  
Amino Acid ◽  
Proteinase Inhibitors ◽  
Sequence Similarity ◽  
Serine Proteinase ◽  
Exchange Chromatography ◽  
Association Constants ◽  
Cathepsin G ◽  
Amino Acid Residues ◽  
Mature Seeds ◽  
Echinocystis Lobata

Two serine proteinase inhibitors (ELTI I and ELTI II) have been isolated from mature seeds of Echinocystis lobata by ammonium sulfate fractionation, methanol precipitation, ion exchange chromatography, affinity chromatography on immobilized anhydrotrypsin and HPLC. ELTI I and ELTI II consist of 33 and 29 amino-acid residues, respectively. The primary structures of these inhibitors are as follows: ELTI I KEEQRVCPRILMRCKRDSDCLAQCTCQQSGFCG ELTI II RVCPRILMRCKRDSDCLAQCTCQQSGFCG The inhibitors show sequence similarity with the squash inhibitor family. ELTI I differs from ELTI II only by the presence of the NH2-terminal tetrapeptide Lys-Glu-Glu-Gln. The association constants (Ka) of ELTI I and ELTI II with bovine-trypsin were determined to be 6.6 x 10(10) M-1, and 3.1 x 10(11) M-1, whereas the association constants of these inhibitors with cathepsin G were 1.2 x 10(7) M-1, and 1.1 x 10(7) M-1, respectively.

Synthesis and conformation of sequential basic polypeptides with branched and linear side chains

1985 ◽  
Vol 50 (12) ◽  
pp. 2925-2936 ◽  
Author(s):  
Štěpánka Štokrová ◽  
Jan Pospíšek ◽  
Jaroslav Šponar ◽  
Karel Bláha
Keyword(s):  
Amino Acid ◽  
Salt Concentration ◽  
Salt Solution ◽  
Theoretical Work ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Cd Spectra ◽  
Low Salt ◽  
Conformational Freedom ◽  
Basic Polypeptides

Polypeptides (Lys-X-Ala)n and (Lys-X-Gly)n in which X represents residues of isoleucine and norleucine, respectively, and polypeptide (Tle-Lys-Ala)n, were synthesized via polymerization of 1-hydroxysuccinimidyl esters of the appropriate tripeptides to complete previously studied series. Circular dichroism (CD) spectra of the respective polymers were measured as a function of pH and salt concentration of the medium. The results were correlated with those obtained previously with the same series containing different amino acid residues at the X-position. The helix forming ability of the polypeptides (Lys-X-Ala)n with linear X side chain was found to be independent of the length. In the series (Lys-X-Gly)n the unordered conformation was the most probable one except (Lys-Ile-Gly)n. This polymer assumed the β conformation even in low salt solution at neutral pH. An agreement with some theoretical work concerned with the restriction of conformational freedom of amino acid residue branching at Cβ atom with our experimental results is evident.

scholarly journals Structure–function studies of human deoxyhypusine synthase: identification of amino acid residues critical for the binding of spermidine and NAD

2001 ◽  
Vol 355 (3) ◽  
pp. 841-849 ◽  
Author(s):  
Chang Hoon LEE ◽  
Patrick Y. UM ◽  
Myung Hee PARK
Keyword(s):  
Crystal Structure ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Binding Sites ◽  
Binding Pocket ◽  
Complete Loss ◽  
Amino Acid Residues ◽  
Deoxyhypusine Synthase ◽  
Positive Identification ◽  
Insight Into

Deoxyhypusine synthase catalyses the first step in the biosynthesis of hypusine [Nε-(4-amino-2-hydroxybutyl)lysine]. The crystal structure of human deoxyhypusine synthase in complex with NAD revealed four NAD-binding sites per enzyme tetramer, and led to a prediction of the spermidine-binding pocket. We have replaced each of the seven amino acid residues at the predicted spermidine-binding site, and eleven residues that contact NAD, on an individual basis with alanine. Of the amino acid residues at the spermidine site, substitution of Asp-243, Trp-327, His-288, Asp-316 or Glu-323 with alanine caused an almost complete loss of spermidine binding and enzyme activity; only the mutation Tyr-305 → Ala showed partial binding and activity. His-288 → Ala was also deficient in terms of binding NAD. NAD binding was significantly reduced in all of the NAD-site mutant enzymes, except for Glu-137 → Ala, which showed a normal binding of NAD, but was totally lacking in spermidine binding. Of the NAD-site mutant enzymes, Asp-342 → Ala, Asp-313 → Ala and Asp-238 → Ala displayed the lowest binding of NAD. These enzymes and His-288Ala also showed a reduced binding of spermidine, presumably because spermidine binding is dependent on NAD. These findings permit the positive identification of amino acid residues critical for binding of spermidine and NAD, and provide a new insight into the complex molecular interactions involved in the deoxyhypusine synthase reaction.

scholarly journals Molecular cloning, gene organization and expression of the human UDP-GalNAc:Neu5Acalpha2-3Galbeta-R beta1,4-N-acetylgalactosaminyltransferase responsible for the biosynthesis of the blood group Sda/Cad antigen: evidence for an unusual extended cytoplasmic domain

2003 ◽  
Vol 373 (2) ◽  
pp. 369-379 ◽  
Author(s):  
Maria-Dolores MONTIEL ◽  
Marie-Ange KRZEWINSKI-RECCHI ◽  
Philippe DELANNOY ◽  
Anne HARDUIN-LEPERS
Keyword(s):  
Amino Acid ◽  
Catalytic Domain ◽  
Human Gene ◽  
Short Form ◽  
Cytoplasmic Tail ◽  
Gene Organization ◽  
Sialic Acid Residue ◽  
Chromosome 17 ◽  
Soluble Form ◽  
Amino Acid Residues

The human Sda antigen is formed through the addition of an N-acetylgalactosamine residue via a β1,4-linkage to a sub-terminal galactose residue substituted with an α2,3-linked sialic acid residue. We have taken advantage of the previously cloned mouse cDNA sequence of the UDP-GalNAc:Neu5Acα2-3Galβ-R β1,4-N-acetylgalactosaminyltransferase (Sda β1,4GalNAc transferase) to screen the human EST and genomic databases and to identify the corresponding human gene. The sequence spans over 35 kb of genomic DNA on chromosome 17 and comprises at least 12 exons. As judged by reverse transcription PCR, the human gene is expressed widely since it is detected in various amounts in almost all cell types studied. Northern blot analysis indicated that five Sda β1,4GalNAc transferase transcripts of 8.8, 6.1, 4.7, 3.8 and 1.65 kb were highly expressed in colon and to a lesser extent in kidney, stomach, ileum and rectum. The complete coding nucleotide sequence was amplified from Caco-2 cells. Interestingly, the alternative use of two first exons, named E1S and E1L, leads to the production of two transcripts. These nucleotide sequences give rise potentially to two proteins of 506 and 566 amino acid residues, identical in their sequence with the exception of their cytoplasmic tail. The short form is highly similar (74% identity) to the mouse enzyme whereas the long form shows an unusual long cytoplasmic tail of 66 amino acid residues that is as yet not described for any other mammalian glycosyltransferase. Upon transient transfection in Cos-7 cells of the common catalytic domain, a soluble form of the protein was obtained, which catalysed the transfer of GalNAc residues to α2,3-sialylated acceptor substrates, to form the GalNAcβ1-4[Neu5Acα2-3]Galβ1-R trisaccharide common to both Sda and Cad antigens.

scholarly journals Determination of three amino acids causing alteration of proteolytic activities of staphylococcal glutamyl endopeptidases

2009 ◽  
Vol 390 (3) ◽  
Author(s):  
Takayuki K. Nemoto ◽  
Toshio Ono ◽  
Yu Shimoyama ◽  
Shigenobu Kimura ◽  
Yuko Ohara-Nemoto
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protease Activity ◽  
Binding Pocket ◽  
Catalytic Triad ◽  
Amino Acid Residues ◽  
Proteolytic Activities ◽  
Hydrophobic Amino Acids ◽  
The Difference

Abstract Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus warneri secrete glutamyl endopeptidases, designated GluV8, GluSE, and GluSW, respectively. The order of their protease activities is GluSE<GluSW<<GluV8. In the present study, we investigated the mechanism that causes these differences. Expression of chimeric proteins between GluV8 and GluSE revealed that the difference is primarily attributed to amino acid residues 170–195, which define the intrinsic protease activity, and additionally to residues 119–169, which affect the proteolytic sensitivity. Among nine substitutions present in residues 170–195 of the three proteases, the substitutions at positions 185, 188, and 189 were responsible for the changes in their activities, and the combination of W185, V188, and P189, which naturally occurs in GluV8, exerts the highest protease activity. W185 and P189 were indispensable for full activity, but V188 could be replaced by hydrophobic amino acids. These three amino acid residues appear to create a substrate-binding pocket together with the catalytic triad and the N-terminal V1, and therefore define the K m values of the proteases. We also describe a method to produce a chimeric form of GluSE and GluV8 that is resistant to proteolysis, and therefore possesses 4-fold higher activity than the wild-type recombinant GluV8.

scholarly journals Bioinformatics Insights Into Microbial Xylanase Protein Sequences

2018 ◽  
Vol 15 (2) ◽  
pp. 275-294
Author(s):  
Deepsikha Anand ◽  
Jeya Nasim ◽  
Sangeeta Yadav ◽  
Dinesh Yadav
Keyword(s):  
Amino Acid ◽  
Phylogenetic Tree ◽  
Catalytic Domain ◽  
Genetic Manipulation ◽  
Protein Sequences ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Molecular Weights ◽  
Aliphatic Index ◽  
Chemical Attributes

Microbial xylanases represents an industrially important group of enzymes associated with hydrolysis of xylan, a major hemicellulosic component of plant cell walls. A total of 122 protein sequences comprising of 58 fungal, 25 bacterial, 19actinomycetes and 20 yeasts xylanaseswere retrieved from NCBI, GenBank databases. These sequences were in-silico characterized for homology,sequence alignment, phylogenetic tree construction, motif assessment and physio-chemical attributes. The amino acid residues ranged from 188 to 362, molecular weights were in the range of 20.3 to 39.7 kDa and pI ranged from 3.93 to 9.69. The aliphatic index revealed comparatively less thermostability and negative GRAVY indicated that xylanasesarehydrophilicirrespective of the source organisms.Several conserved amino acid residues associated with catalytic domain of the enzyme were observed while different microbial sources also revealed few conserved amino acid residues. The comprehensive phylogenetic tree indicatedsevenorganismsspecific,distinct major clusters,designated as A, B, C, D, E, F and G. The MEME based analysis of 10 motifs indicated predominance of motifs specific to GH11 family and one of the motif designated as motif 3 with sequence GTVTSDGGTYDIYTTTRTNAP was found to be present in most of the xylanases irrespective of the sources.Sequence analysis of microbial xylanases provides an opportunity to develop strategies for molecular cloning and expression of xylanase genes and also foridentifying sites for genetic manipulation for developing novel xylanases with desired features as per industrial needs.

scholarly journals Cloning and Sequence Analysis of a New Cellulase Gene Encoding CelK, a Major Cellulosome Component of Clostridium thermocellum: Evidence for Gene Duplication and Recombination

1999 ◽  
Vol 181 (17) ◽  
pp. 5288-5295 ◽  
Author(s):  
Irina Kataeva ◽  
Xin-Liang Li ◽  
Huizhong Chen ◽  
Sang-Ki Choi ◽  
Lars G. Ljungdahl
Keyword(s):  
Amino Acid ◽  
Gene Duplication ◽  
Signal Peptide ◽  
Clostridium Thermocellum ◽  
Catalytic Domain ◽  
Glycosyl Hydrolase ◽  
Size Difference ◽  
Amino Acid Residues ◽  
Cellulase Gene ◽  
Original Activity

ABSTRACT The cellulolytic and hemicellulolytic complex of Clostridium thermocellum, termed cellulosome, consists of up to 26 polypeptides, of which at least 17 have been sequenced. They include 12 cellulases, 3 xylanases, 1 lichenase, and CipA, a scaffolding polypeptide. We report here a new cellulase gene, celK, coding for CelK, a 98-kDa major component of the cellulosome. The gene has an open reading frame (ORF) of 2,685 nucleotides coding for a polypeptide of 895 amino acid residues with a calculated mass of 100,552 Da. A signal peptide of 27 amino acid residues is cut off during secretion, resulting in a mature enzyme of 97,572 Da. The nucleotide sequence is highly similar to that of cbhA(V. V. Zverlov et al., J. Bacteriol. 180:3091–3099, 1998), having an ORF of 3,690 bp coding for the 1,230-amino-acid-residue CbhA of the same bacterium. Homologous regions of the two genes are 86.5 and 84.3% identical without deletion or insertion on the nucleotide and amino acid levels, respectively. Both have domain structures consisting of a signal peptide, a family IV cellulose binding domain (CBD), a family 9 glycosyl hydrolase domain, and a dockerin domain. A striking distinction between the two polypeptides is that there is a 330-amino-acid insertion in CbhA between the catalytic domain and the dockerin domain containing a fibronectin type 3-like domain and family III CBD. This insertion, missing in CelK, is responsible for the size difference between CelK and CbhA. Upstream and downstream flanking sequences of the two genes show no homology. The data indicate thatcelK and cbhA in the genome of C. thermocellum have evolved through gene duplication and recombination of domain coding sequences. celK without a dockerin domain was expressed in Escherichia coli and purified. The enzyme had pH and temperature optima at 6.0 and 65°C, respectively. It hydrolyzedp-nitrophenyl-β-d-cellobioside with aKm and a V max of 1.67 μM and 15.1 U/mg, respectively. Cellobiose was a strong inhibitor of CelK activity, with a Ki of 0.29 mM. The enzyme was thermostable, after 200 h of incubation at 60°C, 97% of the original activity remained. Properties of the enzyme indicated that it is a cellobiohydrolase.

scholarly journals Investigations of Dimethylglycine, Glycine Betaine, and Ectoine Uptake by a Betaine-Carnitine-Choline Transporter Family Transporter with Diverse Substrate Specificity in Vibrio Species

2020 ◽  
Vol 202 (24) ◽  
Author(s):  
Gwendolyn J. Gregory ◽  
Anirudha Dutta ◽  
Vijay Parashar ◽  
E. Fidelma Boyd
Keyword(s):  
Amino Acid ◽  
Glycine Betaine ◽  
Vibrio Parahaemolyticus ◽  
Compatible Solutes ◽  
Binding Pocket ◽  
Compatible Solute ◽  
Amino Acid Residues ◽  
Choline Transporter ◽  
Content Type ◽  
Highly Effective

ABSTRACT Fluctuations in osmolarity are one of the most prevalent stresses to which bacteria must adapt, both hypo- and hyperosmotic conditions. Most bacteria cope with high osmolarity by accumulating compatible solutes (osmolytes) in the cytoplasm to maintain the turgor pressure of the cell. Vibrio parahaemolyticus, a halophile, utilizes at least six compatible solute transporters for the uptake of osmolytes: two ABC family ProU transporters and four betaine-carnitine-choline transporter (BCCT) family transporters. The full range of compatible solutes transported by this species has yet to be determined. Using an osmolyte phenotypic microarray plate for growth analyses, we expanded the known osmolytes used by V. parahaemolyticus to include N,N-dimethylglycine (DMG), among others. Growth pattern analysis of four triple-bccT mutants, possessing only one functional BCCT, indicated that BccT1 (VP1456), BccT2 (VP1723), and BccT3 (VP1905) transported DMG. BccT1 was unusual in that it could take up both compounds with methylated head groups (glycine betaine [GB], choline, and DMG) and cyclic compounds (ectoine and proline). Bioinformatics analysis identified the four coordinating amino acid residues for GB in the BccT1 protein. In silico modeling analysis demonstrated that GB, DMG, and ectoine docked in the same binding pocket in BccT1. Using site-directed mutagenesis, we showed that a strain with all four residues mutated resulted in the loss of uptake of GB, DMG, and ectoine. We showed that three of the four residues were essential for ectoine uptake, whereas only one of the residues was important for GB uptake. Overall, we have demonstrated that DMG is a highly effective compatible solute for Vibrio species and have elucidated the amino acid residues in BccT1 that are important for the coordination of GB, DMG, and ectoine transport. IMPORTANCE Vibrio parahaemolyticus possesses at least six osmolyte transporters, which allow the bacterium to adapt to high-salinity conditions. In this study, we identified several additional osmolytes that were utilized by V. parahaemolyticus. We demonstrated that the compound DMG, which is present in the marine environment, was a highly effective osmolyte for Vibrio species. We determined that DMG is transported via BCCT family carriers, which have not been shown previously to take up this compound. BccT1 was a carrier for GB, DMG, and ectoine, and we identified the amino acid residues essential for the coordination of these compounds. The data suggest that for BccT1, GB is more easily accommodated than ectoine in the transporter binding pocket.

scholarly journals Novel Carbohydrate-Binding Module of β-1,3-Xylanase from a Marine Bacterium, Alcaligenes sp. Strain XY-234

2002 ◽  
Vol 184 (9) ◽  
pp. 2399-2403 ◽  
Author(s):  
Fumiyoshi Okazaki ◽  
Yutaka Tamaru ◽  
Shinnosuke Hashikawa ◽  
Yu-Teh Li ◽  
Toshiyoshi Araki
Keyword(s):  
Amino Acid ◽  
Marine Bacterium ◽  
Catalytic Domain ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Amino Acid Residues ◽  
Glycosyl Hydrolases ◽  
Binding Studies ◽  
Calculated Molecular Mass ◽  
Reading Frame

ABSTRACT A β-1,3-xylanase gene (txyA) from a marine bacterium, Alcaligenes sp. strain XY-234, has been cloned and sequenced. txyA consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da. The domain structure of the β-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus. The recombinant TxyA hydrolyzed β-1,3-xylan but not other polysaccharides such as β-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or β-1,4-mannan. TxyA was capable of binding specifically to β-1,3-xylan. The analysis using truncated TxyA lacking either the N- or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469. Binding studies on the CBM revealed that the Kd and the maximum amount of protein bound to β-1,3-xylan were 4.2 μM and 18.2 μmol/g of β-1,3-xylan, respectively. Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of β-1,3-xylan.

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