(188bq) Truncation and Characterization of the Caffeine N-Demethylase Reductase from Pseudomonas Putida CBB5

Pseudomonas putida CBB5 is a bacterium that has evolved the ability to metabolize caffeine and other methylxanthines to xanthine using a set of five enzymes, NdmABCDE. NdmABC are N­₁-, N₃-, and N₇-specific N-demethylases that are part of the multicomponent Rieske oxygenase family. NdmD is a 65 kDa enzyme that serves as the sole Rieske oxygenase reductase for this set of enzymes and is necessary to transfer electrons from NADP(H) so that the N-demethylation reactions can occur^1-2.

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 FNR_C-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.

1. Summers, R. M.; Louie, T. M.; Yu, C.-L.; Gakhar, L.; Louie, K. C.; Subramanian, M., Novel, Highly Specific N-Demethylases Enable Bacteria To Live on Caffeine and Related Purine Alkaloids. Journal of Bacteriology 2012, 194 (8), 2041-2049.
2. Summers, R. M.; Seffernick, J. L.; Quandt, E. M.; Yu, C. L.; Barrick, J. E.; Subramanian, M. V., Caffeine Junkie: an Unprecedented Glutathione S-Transferase-Dependent Oxygenase Required for Caffeine Degradation by Pseudomonas putida CBB5. Journal of Bacteriology 2013, 195 (17), 3933-3939.