NdmD is unique from other Rieske reductases because it contains an extra Rieske 2Fe-2S cluster at its N-terminal end. This appears to be a fusion from NdmC, which lacks a Rieske domain but contains the N7-demethylation catalytic site. NdmE is a structural protein that holds the NdmC and NdmD proteins together. The C-terminal end of NdmD is similar to other FNRC-type Rieske reductases, with a FAD/FMN binding domain, an NADH binding domain, and a 2Fe-2S plant-type ferredoxin domain1. The hypothesis of this work is that the Rieske 2Fe-2S domain on NdmD is used in conjunction with the Rieske-less NdmC enzyme and structural subunit NdmE to carry out the N7-demethylation of 7-methylxanthine to xanthine,2 but is not required for activity with the NdmA and NdmB enzymes, which contain their own Rieske 2Fe-2S clusters.
In order to test this hypothesis, a truncated version of NdmD lacking the N-terminal Rieske cluster was cloned into the pET28a(+) E. coli expression plasmid with an N-terminal hexahistidine tag and named NdmDP1. NdmDP1 was expressed and purified from E. coli cultures using a nickel affinity column. Cytochrome c reductase and N-demethylase enzymatic assays1 were performed using the purified NdmDP1 enzyme in conjunction with NdmA and NdmB. Results indicate that the N-terminal Rieske 2Fe-2S cluster are not necessary for activity of NdmA or NdmB, but is required for NdmC activity.