MD and QM/MM study on catalytic mechanism of a FAD-dependent enzyme ORF36: for nitro sugar biosynthesis.

The catalytic mechanism of a FAD-dependent nitrososynthase (ORF36) was studied with molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) methods. Residues Leu160 and Phe374 play an important role during the FAD binding with ORF36. Similar phenylalanine/leucine pair was found in the other two enzymes of this family. For the second oxidation step of ORF36 toward thymidine diphosphate-l-epi-vancosamine, three elementary catalytic steps were found: a hydroxylation step, a hydrogen back-transfer step and a hydroxyl group elimination step. The hydroxylation step is found to be the rate-determining step with an energy barrier of 26.3kcal/mol under the B3LYP/cc-pVTZ//CHARMM22 level. Two possible pathways for the second oxidation step are carefully investigated. Our simulations indicate that an oxygen atom from the coenzyme FADHOOH is inserted into the product. In addition, the electrostatic influence of 17 individual residues and five neighboring water molecules on the rate-determining step was estimated. The results indicate that groups Gly132/Ala133/Leu134, Met375/Gln376 and a water fence play a key role in facilitating the rate-determining step. On the other hand, residues Leu160, Val161 and Ser162 are found to be critical to suppress the rate-determining step. Our results lead to further understanding of the detailed catalytic pathways for nitro sugar biosynthesis.