Background
The development of high-throughput genome squencing allowed proteins to be sequenced more quickly than their structures could be solved. In an effort to close this gap, in 2000, the National Institutes of Health launched the 15-year Protein Structure Initiative. Solved structures are deposited in the Protein Data Bank, but many proteins, such as YxiM (PDB ID: 2O14), remain functionally uncharacterized. YxiM originates from Bacillus subtilis, a ubiquitous bacterial species that dwells in soil and gastrointestinal tracts. YxiM is 375 amino acids in length and its molecular weight is 41.8 kDa. It appears to have two domains: one dominated by α-helices, and one by β-sheets.
Methods
We first transformed DH5α Competent E. coli cells with the plasmid to create more plasmid.
Then, we expressed
Results
This is the .
Discussion
YxiM is a previously uncharacterized protein whose crystal structure has been solved and deposited in the PDB. On the basis of protein sequence and structural analysis in silico and functional assays in vitro, we conclude that YxiM is an esterase. There is some disagreement regarding the definitions of esterases and lipases. We consider lipases to be a subclass of esterases; lipases specifically hydrolyze lipids, whereas esterases hydrolyze ester bonds in general.
Sequence analysis with BLAST and PFam suggests that YxiM is a GDSL-like lipase, a type of lipase that demonstrates broad substrate specificity due to a flexible structure. Proteins with the highest sequence homology to YxiM are multifunctional hydrolases and show both esterase and protease activity. PyMOL shows that these hits also align well with the 3D structure of the α-helix domain of YxiM. Almost all the top structural hits in Dali are esterases as well, and ProMOL shows that the active site of YxiM most resembles one of an esterase or protease. The same catalytic triad (S171, D339, H342) is implicated in both protease and esterase activity, suggesting YxiM could be a multifunctional hydrolase. The catalytic motif of the esterase 1BWR aligns particularly well with YxiM.
Given that all in silico tests suggest that YxiM is an esterase, we tested YxiM for esterase activity in vitro. Protein expression and purification were successful. YxiM showed esterase activity on 4-nitrophenyl butyrate, as absorbance increased during the assay. Our Lineweaver-Burk plot of YxiM esterase activity is linear, which is typical of enzymes.
For future studies, we can further confirm the esterase activity of YxiM by performing mutagenesis on the putative catalytic motif. Our data also suggest a potential protease functionality for YxiM. To test this, we could perform protease assays as well. To further study enzyme kinetics, we need to relate absorbance with concentration of protein. We can achieve this by performing a Bradford protein assay to compute the extinction coefficient.
Future Directions
