Proposed amino acid sequence and the 1.63 Å X-ray crystal structure of a plant cysteine protease, ervatamin B: Some insights into the structural basis of its stability and substrate specificity

2003 ◽  
Vol 51 (4) ◽  
pp. 489-497 ◽  
Author(s):  
Sampa Biswas ◽  
Chandana Chakrabarti ◽  
Suman Kundu ◽  
Medicherla V. Jagannadham ◽  
Jiban K. Dattagupta
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Specificity ◽  
Cysteine Protease ◽  
Structural Basis ◽  
X Ray

Structural insights into the substrate specificity of a glycoside hydrolase family 5 lichenase from Caldicellulosiruptor sp. F32

2017 ◽  
Vol 474 (20) ◽  
pp. 3373-3389 ◽  
Author(s):  
Dong-Dong Meng ◽  
Xi Liu ◽  
Sheng Dong ◽  
Ye-Fei Wang ◽  
Xiao-Qing Ma ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Glycoside Hydrolase ◽  
Complex Structure ◽  
Dynamics Simulation ◽  
Structural Basis ◽  
Glycoside Hydrolase Family ◽  
Substrate Selectivity ◽  
Glucose Residue ◽  
Structural Insights

Glycoside hydrolase (GH) family 5 is one of the largest GH families with various GH activities including lichenase, but the structural basis of the GH5 lichenase activity is still unknown. A novel thermostable lichenase F32EG5 belonging to GH5 was identified from an extremely thermophilic bacterium Caldicellulosiruptor sp. F32. F32EG5 is a bi-functional cellulose and a lichenan-degrading enzyme, and exhibited a high activity on β-1,3-1,4-glucan but side activity on cellulose. Thin-layer chromatography and NMR analyses indicated that F32EG5 cleaved the β-1,4 linkage or the β-1,3 linkage while a 4-O-substitued glucose residue linked to a glucose residue through a β-1,3 linkage, which is completely different from extensively studied GH16 lichenase that catalyses strict endo-hydrolysis of the β-1,4-glycosidic linkage adjacent to a 3-O-substitued glucose residue in the mixed-linked β-glucans. The crystal structure of F32EG5 was determined to 2.8 Å resolution, and the crystal structure of the complex of F32EG5 E193Q mutant and cellotetraose was determined to 1.7 Å resolution, which revealed that the exit subsites of substrate-binding sites contribute to both thermostability and substrate specificity of F32EG5. The sugar chain showed a sharp bend in the complex structure, suggesting that a substrate cleft fitting to the bent sugar chains in lichenan is a common feature of GH5 lichenases. The mechanism of thermostability and substrate selectivity of F32EG5 was further demonstrated by molecular dynamics simulation and site-directed mutagenesis. These results provide biochemical and structural insights into thermostability and substrate selectivity of GH5 lichenases, which have potential in industrial processes.

The amino acid sequence of yeast phosphoglycerate mutase

1982 ◽  
Vol 215 (1198) ◽  
pp. 19-44 ◽  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Proteolytic Enzymes ◽  
Phosphoglycerate Mutase ◽  
Crystallographic Structure ◽  
X Ray ◽  
C Terminus ◽  
Detailed Interpretation ◽  
High Degree

The complete amino acid sequence of yeast phosphoglycerate mutase comprising 241 residues has been determined. The sequence was deduced from the two cyanogen bromide fragments, and from the peptides derived from these fragments after digestion by a number of proteolytic enzymes. Determination of this sequence now allows a detailed interpretation of the existing high-resolution X-ray crystallographic structure. A comparison of the sequence reported here with the sequences of peptides from phosphoglycerate mutases from other species, and with the sequence of erythrocyte diphosphoglycerate mutase, indicates that these enzymes have a high degree of structural homology. Autolysis of phosphoglycerate mutase by yeast extracts leads to the complete loss of mutase activity, and the formation of electrophoretically distinguishable forms (R. Sasaki, E. Sugimoto & H. Chiba, Archs Biochem. Biophys. 115, 53-61 (1966)). It is apparent from the amino acid sequence that these changes are due to the loss of an 8─12 residue peptide from the C-terminus.

Amino acid sequence and some properties of phytolacain G, a cysteine protease from growing fruit of pokeweed, Phytolacca americana

2000 ◽  
Vol 1523 (2-3) ◽  
pp. 254-260 ◽  
Author(s):  
Tetsuya Uchikoba ◽  
Kazunari Arima ◽  
Hiroo Yonezawa ◽  
Masayuki Shimada ◽  
Makoto Kaneda
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cysteine Protease ◽  
Phytolacca Americana

Purification, characterization, gene cloning and preliminary X-ray data of the exo-inulinase from Aspergillus awamori

2002 ◽  
Vol 362 (1) ◽  
pp. 131-135 ◽  
Author(s):  
Michael ARAND ◽  
Alexander M. GOLUBEV ◽  
J. R. Brandao NETO ◽  
Igor POLIKARPOV ◽  
R. WATTIEZ ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Solid Phase ◽  
Aspergillus Awamori ◽  
Glycoside Hydrolase Family ◽  
Orthorhombic Space Group ◽  
Ph Optimum ◽  
X Ray ◽  
Cell Parameters ◽  
Hydrolysis Of

Extracellular exo-inulinase has been isolated from a solid-phase culture of the filamentous fungus Aspergillus awamori var. 2250. The apparent molecular mass of the monomer enzyme was 69±1kDa, with a pI of 4.4 and a pH optimum of 4.5. The enzyme hydrolysed the β-(2 → 1)-fructan (inulin) and β-(2 → 6)-fructan (levan) via exo-cleavage, releasing fructose. The values for the Michaelis constants Km and Vmax in the hydrolysis of inulin were 0.003±0.0001mM and 175±5μmol·min−1·mg−1. The same parameters in the hydrolysis of levan were 2.08±0.04mg/ml and 1.2±0.02μmol/min per mg, respectively. The gene and cDNA encoding the A. awamori exo-inulinase were cloned and sequenced. The amino acid sequence indicated that the protein belongs to glycoside hydrolase family 32. A surprisingly high similarity was found to fructosyltransferase from Aspergillus foetidus (90.7% on the level of the amino acid sequence), despite the fact that the latter enzyme is unable to hydrolyse inulin and levan. Crystals of the native exo-inulinase were obtained and found to belong to the orthorhombic space group P212121 with cell parameters a = 64.726 Å (1Å = 0.1 nm), b = 82.041 Å and c = 136.075 Å. Crystals diffracted beyond 1.54 Å, and useful X-ray data were collected to a resolution of 1.73 Å.

scholarly journals Genetic variability in cysteine protease genes ofHaemonchus contortus

Parasitology ◽  
2004 ◽  
Vol 128 (5) ◽  
pp. 549-559 ◽  
Author(s):  
A. RUIZ ◽  
J. M. MOLINA ◽  
A. NJUE ◽  
R. K. PRICHARD
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Genetic Variability ◽  
Amino Acid Sequence ◽  
Cysteine Protease ◽  
Haemonchus Contortus ◽  
Significant Variation ◽  
Nucleotide Diversity ◽  
Cysteine Proteases ◽  
Different Strains

To increase the existent genetic variability in cysteine proteases, a polymorphism study was performed inHaemonchus contortusby comparing 2 different strains of the parasite: North American (NA) and Spanish (SP) strains. For this purpose, the polymorphism of 5 previously reported genes (AC-1,AC-3,AC-4,AC-5andGCP-7) were analysed by PCR–SSCP and sequencing procedures. Based on the SSCP results, a total of 20 different alleles were identified for the 5lociassessed. Exceptlocus AC-5, all thelociwere polymorphic.Loci AC-1,AC-3,AC-4andGCP-7showed 5, 8, 2 and 4 alleles, respectively. The allelic frequencies ranged from 0·0070 to 0·8560 and were significantly different between strains. In addition, nucleotide diversity analyses showed a significant variation within and between strains. The variations in the nucleotide sequence of the different alleles were translated in some cases into changes in the amino acid sequence. Evidence of genetic variability in cysteine proteases from two different strains ofH. contortusfor the same set of genes had not been previously reported.

scholarly journals Structural Basis for Imipenem Inhibition of Class C β-Lactamases

2002 ◽  
Vol 46 (12) ◽  
pp. 3978-3980 ◽  
Author(s):  
Beth M. Beadle ◽  
Brian K. Shoichet
Keyword(s):  
Crystal Structure ◽  
Carbonyl Oxygen ◽  
Structural Basis ◽  
Enzyme Complex ◽  
X Ray ◽  
Class A ◽  
Lactam Carbonyl ◽  
Class C ◽  
Expected Position

ABSTRACT To determine how imipenem inhibits the class C β-lactamase AmpC, the X-ray crystal structure of the acyl-enzyme complex was determined to a resolution of 1.80 Å. In the complex, the lactam carbonyl oxygen of imipenem has flipped by approximately 180° compared to its expected position; the electrophilic acyl center is thus displaced from the point of hydrolytic attack. This conformation resembles that of imipenem bound to the class A enzyme TEM-1 but is different from that of moxalactam bound to AmpC.

scholarly journals The crystal structure of a xyloglucan-specific endo-β-1,4-glucanase from Geotrichum sp. M128 xyloglucanase reveals a key amino acid residue for substrate specificity

FEBS Journal ◽  
2009 ◽  
Vol 276 (18) ◽  
pp. 5094-5100 ◽  
Author(s):  
Katsuro Yaoi ◽  
Hidemasa Kondo ◽  
Ayako Hiyoshi ◽  
Natsuko Noro ◽  
Hiroshi Sugimoto ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Amino Acid Residue
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