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Variants of an active site histidine in Rv0045c esterase from M. tuberculosis (580.3)
Author(s) -
Lukowski Jessica,
McKary Magy,
Hoops Geoffrey,
Johnson R. Jeremy
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.580.3
Subject(s) - histidine , asparagine , biochemistry , tyrosine , esterase , chemistry , serine , active site , phenylalanine , amino acid , residue (chemistry) , enzyme , tryptophan , site directed mutagenesis , mycobacterium tuberculosis , valine , biology , tuberculosis , medicine , pathology , mutant , gene
Mycobacterium tuberculosis , from which the Rv0045c protein is isolated, is the most causative bacterium leading to tuberculosis. Histidine (H187), within the active site of this esterase, was the residue of interest in this project. The histidine was mutated to ten different amino acid residues (aspartate, asparagine, glycine, lysine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine) and the catalytic efficiency of the enzyme variants was determined. Variant proteins were produced through site‐directed mutagenesis, overexpression in E. coli cells, and purification using a Ni 2+ affinity resin. Evidence from differential scanning fluorimetry (DSF) revealed no significant differences in the thermal stability of the variant proteins. Michaelis‐Menton kinetics revealed that most of the variant proteins showed no difference in catalytic activity from the wildtype protein. The tyrosine variant increased the protein’s overall kinetic efficiency. A sequence alignment with esterases from similar organisms showed that a tyrosine residue is almost as common as the histidine residue present in Rv0045c. Grant Funding Source : National Science Foundation