Arg²³⁵ is an essential catalytic residue of Bacillus pumilus DKS1 pectate lyase to degum ramie fibre.
After 24 h of incubation with only purified pectate lyase isolated from Bacillus pumilus DKS1 (EF467045), the weight loss of the ramie fibre was found to be 25%. To know the catalytic residue of pectate lyase the pel gene encoding a pectate lyase from the strain Bacillus pumilus DKS1 was cloned in E. coli XL1Blue and expressed in E. coli BL21 (DE3) pLysS. The pel gene was sequenced and showed 1032 bp length. After purification using CM-Sepharose the enzyme showed molecular weight of 35 kDa and maximal enzymatic activity was observed at 60°C and a pH range of 8.5-9.0. Both Ca²(+) and Mn²(+) ions were required for activity on Na-pectate salt substrates, while the enzyme was strongly inhibited by Zn²(+) and EDTA. The deduced nucleotide sequence of the DKS1 pectate lyase (EU652988) showed 90% homology to pectate lyases from Bacillus pumilus SAFR-032 (CP000813). The 3D structure as well as the catalytic residues was predicted using EasyPred software and Catalytic Site Atlas (CSA), respectively. Site directed mutagenesis confirmed that arginine is an essential catalytic residue of DKS1 pectate lyase.
Basu
Snehasish
S
Bose Institute, Kolkata, India.
Roy
Arunava
A
Ghosh
Abhrajyoti
A
Bera
Amit
A
Chattopadhyay
Dhrubajyoti
D
Chakrabarti
Krishanu
K
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D032402:Boehmeria / Q000737:chemistry*; D020134:Catalytic Domain; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010368:Pectins / Q000737:chemistry; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D013379:Substrate Specificity
D001426:Bacterial Proteins; D010368:Pectins; D001120:Arginine; D011133:Polysaccharide-Lyases; C023911:pectate lyase