X-RAY CRYSTAL STRUCTURES OF DIHYDROMETHANOPTERIN REDUCTASE A: CHARACTERIZATION OF SUBSTRATE BINDING AND LOOP MOVEMENTS USING COMPUTATIONAL MODELING

The facultative methylotroph Methylobacterium extorquens AM1 uses tetrahydromethanopterin (H4MPT) as a one-carbon carrying cofactor to grow on single-carbon compounds. The last step of H4MPT sidechain biosynthesis involves reducing dihydromethanopterin (H2MPT) to H4MPT. This reaction is catalyzed by dihydromethanopterin reductase A (DmrA). It is hypothesized that DmrA has evolved from an ancestral bacterial dihydrofolate reductase (DHFR). Previous kinetic assays have revealed that DmrA has shown activity in the presence of dihydrofolate (DHF) reducing it to tetrahydrofolate (THF). In this study, the crystal structure of DmrA is used to uncover a deeper understanding of the interactions of H2MPT with DmrA and the roles of loop conformational changes in enzymatic activity. From the sequence alignment of DmrA and DHFR from Escherichia coli, there is a replacement of two methionines from the E. coli Met20 loop with leucines. Moreover, there is an extension of this loop to include the previously named DmrA loop. The DmrA crystal structure contains seven beta-strands and six alpha-helices. Computational docking has found that H2MPT forms hydrogen bonds with both Glu25 and Gly26, while DHF interacts only with Glu25. Moreover, molecular dynamics predicted that the residue 11-27 loop interacts with the pterin ring of H2MPT to bring it closer to NADPH for reduction during the catalytic process. Therefore, the loop movement is hypothesized to be crucial for the reduction of H2MPT to H4MPT