Role of conserved active site tryptophan-101 in functional activity and stability of phosphoserine aminotransferase from an enteric human parasite.
Site-directed mutagenesis study was performed to elucidate the role of conserved tryptophan-101 present at the active site of phosphoserine aminotransferase from an enteric human parasite Entamoeba                         histolytica. Fluorescence resonance energy transfer and molecular dynamic simulation show that the indole ring of Trp101 stacks with the cofactor PLP. Loss of enzymatic activity and PLP polarization values suggest that Trp101 plays a major role in maintaining a defined PLP microenvironment essentially required for optimal enzymatic activity. Studies on W101F, W101H and W101A mutants show that only the indole ring of the conserved Trp101 forms most favorable stacking interaction with the pyridine ring of the cofactor PLP. Protein stability was compromised on substitution of Trp101 with Phe/His/Ala amino acids. A difference in conformational free energy of 1.65 kcal mol(-1) was observed between WT-protein and W101A mutant.
Division of Molecular and Structural Biology, CSIR-Central Drug Research Institute, Lucknow, 226001, India. vibhor6strings@rediffmail.com
Vibhor
V
Mishra
Ashutosh
A
Kumar
Vahab
V
Ali
Tomoyoshi
T
Nozaki
Kam Y J
KY
Zhang
Vinod
V
Bhakuni
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004748:Entamoeba histolytica / Q000166:cytology / Q000201:enzymology*; D004795:Enzyme Stability; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011732:Pyridoxal Phosphate / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry*
D011732:Pyridoxal Phosphate; D014364:Tryptophan; D000637:Transaminases; C022393:phosphoserine aminotransferase