Substrate Specificity and Cofactor Preference of Mathanosarcina mazei Rib7 Reductase Involved in Riboflavin Biosynthesis
| Autor: | Ting-Hao Chang, 張庭皓 |
|---|---|
| Rok vydání: | 2009 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 97 During riboflavin biosynthesis, archaeal and fungal Rib7s catalyze the reductive transformation of the ribosyl group into ribitol. We report here several crystal structures of Mathanosarcina mazei Rib7 at 2.25Å resolution, in which three crystal forms were obtained with high solvent contents of 60-80 %. Due to crystal packing in the R3 form, clear electron density was observed for the 12 additional histidine-tagged vector residues (MRGSH6GS) at the N terminus and residues 225-228 at the C terminus. These are the first dimeric Rib7 structures, in which the dimeric interface includes extensive hydrophobic patches and 24 direct hydrogen bonds. MmRib7 displays high structural homology to eubacterial RibG and dihydrofolate reductase. Unexpectedly, MmRib7 shares a similar inter-subunit orientation to the Thermotoga maritime dihydrofolate reductase. In addition, the substrate-binding residues of M. mazei Rib7 were predicted: no detectable activity was observed in the D33N, D33K, and E156Q mutants, while the N9A, S29A, D33A, and D57N variants displayed a decreased activity by 2-3 orders of magnitude compared with the wild-type enzyme. Crystal structures of the D33A and D33N variants suggest that Asp33 in M. mazei Rib7 ensures the specific recognition of the substrate amino group. On the other hand, the eubacterial RibGs utilize a conserved lysine to recognize the substrate carbonyl group, Lys151 in BsRibG. Replacement of the conserved lysine with aspartate or glutamate cannot switch the substrate specificity in eubacteria, versus in archaea. Moreover, MmRib7 structures showed an endogenous cofactor NADP, in which the 2’-phosphate group interacts with Ser88 and Arg91. Replacement of Ser88 with glutamate eliminates the endogenous NADP binding and reduces the NADPH-, but not NADH-dependent reductase activity. The mutant activity decreases rapidly with time, and the protein was less heat resistant than wild type. This demonstrates that like the most common human enzymopathy, glucose-6-phosphate dehydrogenase, the tight bound NADP secures the long-term structural stability with attainment of the active conformation. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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