Dissertation Defense: Bok-Eum Choi
Candidate Name: Bok-Eum Choi
Advisor: David C.H. Yang, Ph.D.
Title: Mechanistic Insights of Reactions Catalyzed by OMPB Lysine Methyltransferases
Protein lysine methylation of rickettsial OmpB (outer membrane protein B) has been implicated in the virulence of the typhus group of Rickettsia. N- and O-methylations have also been observed in virulence-relevant outer membrane proteins from several pathogenic bacteria that cause typhus, leptospirosis, tuberculosis and anaplasmosis. To investigate the regulatory and catalytic mechanism of rickettsial OmpB methylation, our lab has bioinformatically identified, and biochemically characterized, including crystallographic analysis for two families of rickettsial protein lysine methyltransferases, PKMT1 and PKMT2. PKMT1 catalyzes predominantly monomethylation while PKMT2 catalyzes mainly trimethylation of OmpB. These enzymes are unique in that they catalyze OmpB lysyl trimethylation and monomethylation at multiple sites with diverse amino acid sequence contexts. The mechanisms of OmpB methylation catalyzed by these methyltransferases are investigated using integrated liquid chromatography-tandem mass spectrometry, enzyme-structure guided site-directed mutagenesis, and steady state enzyme kinetic analyses. Three distinctive structural elements detected in these methyltransferases are the amino-terminal extension in PKMT1, the elongated loop in PKMT2, and the substitutions of three Tyr residues in PKMT1 with Phe in PKMT2. Alteration of these elements only changes the magnitude of their KM and/or kcat, without affecting their catalytic functions. H222 and E223 in the active site of PKMT2 stabilize the transition state intermediate via a hydrogen bond relay with targeting lysyl residue. Furthermore, PKMT2 catalyzes OmpB trimethylation via a processive mechanism in which the methylated intermediates remain enzyme-bound. Both PKMT1 and PKMT2 catalyze methylation of multiple lysyl residues via an ordered sequential mechanism in which both enzymes initiate their methylation at the same high-affinity sites. However, PKMT1 exhibits much higher catalytic activity than PKMT2. It exerts a synergistic effect on the catalytic activity of PKMT2, mediated by coupling the methylation substrates of PKMT1 and PKMT2. Crystal structure of MT-AdoMet-OmpB fragment complex would reveal enzyme-substrate interacting sites, to facilitate the elucidation of the catalytic mechanism. To this end, a short and active peptide fragment, patents after part of the OmpB sequence has been designed and synthesized. This mechanistic investigation of rickettsial methyltransferases would likely yield new insight to facilitate the development of better vaccines, diagnostic tools, and therapeutic drugs.
Friday, May 4 at 9:30am to 11:30am
Regents Hall, 221
3700 O St. NW