Abstract:
Serine hydrolases are one of the largest and most diverse enzyme families. The members of this family play important roles in almost all physiological processes and many are therapeutic targets. Yet many of their members remain unannotated. In this work, I have annotated one such orphan serine hydrolase ABHD14B (alpha-beta hydrolase domain containing protein 14B). This enzyme was first discovered as an interactor of the HAT (histone acetyl transferase) domain of the general transcription factor, TF2D (transcription factor 2D) using a yeast two hybrid cross. On solving its structure, it was found to have a catalytic triad (Serine-Histidine- Aspartate) in its active site. It was also found to have a canonical “alpha-beta hydrolase domain” from where it got its name and it’s classification as a metabolic serine hydrolase. To study this enzyme in greater detail, I recombinantly expressed and purified the wild type (WT) and S111A mutant of this protein using E.coli BL21 (DE3) cells and showed that the S111A mutant was inactive. Using para-nitrophenol (pNP) analogs of different chain lengths as surrogate substrates, I found that this enzyme has a strict preference for shorter substrates and hydrolysed the acetate analog much more efficiently than any of the longer chain length analogs. Using LC-MC methods, I was able to show that this enzyme can transfer the cleaved acetate group from pNP-acetate to a molecule of Coenzyme A (CoA) making acetyl-CoA (AcCoA). I was further able to show that it can also cleave the acetyl group from acetylated lysines on proteins and peptides and form AcCoA. Knocking down this protein in HEK293T cells resulted in lowered AcCoA levels and an increase in protein lysine acetylation. Taking these results together, I annotated ABHD14B as a novel lysine deacetylase. I call it novel because it’s mechanism of action is very different from that of other known lysine deacetylases, namely HDACs (histone deacetylases) and sirtuins. Using PSI-BLASTS and multiple sequence alignments, I was able to discover that ABHD14B is only found in Chordates and that 40% of its residues were conserved. I made point mutations of select residues and studied them biochemically. Mutating residues K141 and S75 showed changes in enzyme biochemistry and they emerged as sites for potential post-translational modifications that could cause allosteric regulation of activity. Lastly, I studied the protein expression and cellular localisation of this protein in fed vs fasted mouse livers to see whether a fasted state was a trigger for ABHD14B’s activity in vivo. A lot still is unknown about this protein and going ahead, its specific protein substrates and its activity in vivo need to be studied.
