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B6db references: 30826314

type Journal Article
authors Israr M, Lv G, Xu S, Li Y, Ding S, Zhao B, Ju J
title Biochemical characterization and mutational analysis of alanine racemase from Clostridium perfringens
journal J Biosci Bioeng
Activity 5.1.1.1
Family 5.1.1.1.a
sel selected
ui 30826314
year (2019)
volume 128
number 2
pages 149-155
 
keywords Alanine racemase; Clostridium perfringens; Dimer interface; Entryway corridor; Site-directed mutagenesis
abstract Clostridium perfringens is a gram-positive, anaerobic, pathogenic bacterium that can cause a wide range of diseases in humans, poultry and agriculturally important livestock. A pyridoxal-5-phosphate-dependent alanine racemase with a function in the racemization of d- and l-alanine is an attractive drug target for C. perfringens and other pathogens due to its absence in animals and humans. In this study alanine racemase from C. perfringens (CPAlr) was successfully expressed and purified in Escherichia coli and biochemically characterized. The purified CPAlr protein was a dimeric PLP-dependent enzyme with high substrate specificity. The optimal racemization temperature and pH were 40°C and 8.0, respectively. The kinetic parameters Km and kcat of CPAlr, determined by HPLC at 40°C were 19.1 mM and 17.2 s-1 for l-alanine, and 10.5 mM and 8.7 s-1 for d-alanine, respectively. Gel filtration chromatographic analysis showed that the molecular weight of mutant Y359A was close to monomeric form, suggesting that the inner layer residue Tyr359 might play an essential role in dimer-formation. Furthermore, the mutation at residues Asp171 and Tyr359 resulted in a dramatic increase in Km value and/or decreased in kcat value, indicating that the middle and inner layer residues Asp171 and Tyr359 of CPAlr might have the key role in substrate binding, catalytic activity or oligomerization state through the hydrogen-bonding interaction with the pentagonal ring waters and/or PLP cofactor.
last changed 2019/12/19 14:19

B6db references