| Summary: | 博士 === 國立陽明大學 === 生化暨分子生物研究所 === 97 === Riboflavin, the universal precursor of flavocoenzymes for a variety of essential cellular processes, is biosynthesized in plants and many microorganisms, but not in animals. Therefore, proteins involved in riboflavin biosynthesis have become attractive targets for antimicrobial drug design. The biosynthetic pathway in most eubacteria contains a bifunctional protein such as Bacillus subtilis RibG (BsRibG), which catalyzes the pyrimidine deamination and the ribosyl reduction. In fungi and some archaea, the reduction is first catalyzed by Rib7 and then the deamination by Rib2. In this thesis, we focus on studying structure and function of BsRibG, Mathanosarcina mazei Rib7 (MmRib7) and yeast Rib2. Together with site-directed mutagenesis and activity assay, the catalytic mechanisms and the distinct substrate recognition have been addressed.
BsRibG consists of an N-terminal deaminase and a C-terminal reductase. The deaminase in complex with AROPP displays that upon the product binding, significant conformational changes are induced in two loops moving toward for interaction with the ribosyl and phosphate groups. Like cytidine and blasticidin S deaminases, the carbonyl moiety coordinates the zinc ion with significantly geometric distortions, revealing a possible common product binding mode in the superfamily. The substrate-binding residues of yeast Rib2 involved in a different biosynthetic pathway are predicted: substitution of Glu545 and Arg518 with alanine eliminates the deaminase activity. Furthermore, a detailed structural comparison reveals a putative amino-binding hole, in which the two consecutive carbonyl backbones located before the PCXXC motif may interact with the amino groups of the substrate and intermediate. This is conserved in most of the superfamily members including the RNA (DNA)-editing deaminases.
For the reductases, the complex structure of BsRibG with the substrate unexpectedly showed a ribitylimino intermediate bound at the active site, and hence suggested that the ribosyl reduction occurs through a Schiff base pathway. Lys151 seems to have evolved to ensure specific recognition of the deaminase product rather than the substrate. Glu290, instead of the previously proposed Asp199, would seem to assist in the proton transfer in the reduction reaction. In addition, the MmRib7 structure displays the first dimeric Rib7 structure with an interface of ~3600 Å2. The crystal structures suggest that Lys151 in BsRibG, while Asp33 in MmRib7, is the key residue for substrate recognition. Interestingly, an endogenous NADP+ was observed in crystal. This cofactor forms more extensive interactions with MmRib7 than BsRibG. Finally, a detailed comparison reveals that the reductases and the pharmaceutically important enzyme, dihydrofolate reductase involved in the riboflavin and folate biosyntheses, share strong conservation of the core structure, cofactor binding, catalytic mechanism, even the substrate binding architecture.
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