Vitamin K is a prokaryotic electron transporter and essential in many gram-positive pathogenic microorganisms. The absence of vitamin K biosynthesis in mammals makes the pathway enzymes attractive targets for development of antibiotics towards the pathogens. To guide the drug discovery, detailed understanding of the catalytic mechanism of the enzyme target is required. In this thesis, we have elucidated mechanistic problems of two enzymes, DHNA-CoA synthase (MenB) and SHCHC synthase (MenH) in the menaquinone (vitamin K2) biosynthesis using structural and biological methods. The first two chapters present the study of MenB. We have firstly investigated the involvement of a bicarbonate cofactor in the crystal structure of the E. coli MenB and discovered a specific binding site fo...[ Read more ]

Vitamin K is a prokaryotic electron transporter and essential in many gram-positive pathogenic microorganisms. The absence of vitamin K biosynthesis in mammals makes the pathway enzymes attractive targets for development of antibiotics towards the pathogens. To guide the drug discovery, detailed understanding of the catalytic mechanism of the enzyme target is required. In this thesis, we have elucidated mechanistic problems of two enzymes, DHNA-CoA synthase (MenB) and SHCHC synthase (MenH) in the menaquinone (vitamin K2) biosynthesis using structural and biological methods. The first two chapters present the study of MenB. We have firstly investigated the involvement of a bicarbonate cofactor in the crystal structure of the E. coli MenB and discovered a specific binding site for bicarbonate close to the MenB active site. Structure comparison and superposition also revealed the catalytic role of the ion in the MenB-catalytzed reaction. The results support our previously proposed classification of the MenB family and provide structural evidence to bicarbonate dependence of the type I MenB. We then investigated the specific interaction between MenB and its substrate analog 1-HNA-CoA through co-crystallization. The crystal structure showed two significant structural changes of MenB after bound to the ligand. The result suggests an induced-fit mechanism of MenB catalysis, whereas the conformational change may relate to substrate recognition and enzyme specificity. The MenH structural and mechanic studies are summarized in the latter two chapters. MenH is a typical α/β hydrolase fold enzyme but does not use the classic nucleophilic mechanism for catalysis. To study the uncanonical function of MenH, we have solved the ligand-free and product-complexed crystal structures of MenH in Chapter 4. An open to close conformational change of the mobile cap domain has been identified that tightly couples with formation of the active catalytic triad in MenH. This conformational change controlled by binding of the ligand accounts for the non-canonical activity of MenH catalytic triad. Rapid kinetics and NMR studies have also revealed that MenH adopts a conformational selection mechanism. Furthermore, we have also undertaken mutagenesis study on a conserved hydrophobic patch on the cap-loop of MenH to elucidate its role in the open-close conformational change as well as MenH catalysis. The cap-loop was shown to participate in the activation of catalytic triad and precisely binding of the substrate via modulating folding and motion of the cap domain. These results not only solve the mechanistic problem of the two enzymes, MenB and MenH, but also provide a knowledge base for future development of high potential inhibitors toward these enzymes as antibiotics targeting the menaquinone biosynthetic pathway.