**3.1.2 Nucleotide preference of bacterial DHDPR**

Pyridine nucleotide-dependent dehydrogenases typically have a strong preference for either NADPH or NADH as co-factors (Cirilli et al., 2003; Pearce et al., 2008; Reddy et al., 1996). In most cases dual-cofactor enzymes preferentially utilise NADPH over NADH. In light of this observation, there has been significant interest in studying the molecular basis of nucleotide preference. All NAD-dependent dehydrogenases contain the consensus sequence GXGXXG or GXXGXXG and conserved acidic amino acids 20-30 residues downstream of this glycine rich region (Dommaraju et al., 2011). The main chain nitrogen of the second residue (X) in the consensus sequence interacts with this conserved acidic residue. *E. coli* DHDPR has an unusual pyridine nucleotide specificity, exhibiting only a modest selectivity for its nucleotides. Kinetic studies show that *E. coli* DHDPR utilises NADH only slightly more efficiently than NADPH (Reddy et al., 1996). This is consistent with the observation that the binding affinity of *E. coli* DHDPR to NADH (*K*D = 0.26 M) is stronger than that of NADPH (*K*D = 1.8 M) (Reddy et al., 1996). Structural studies of *E. coli* DHDPR show the existence of hydrogen bonds between the side-chain of the acidic residue Glu38 and that of the O3' of the adenine ribose of NADH. It is hypothesised that the basic residue Arg39, also found in the nucleotide binding pocket, can interact with the negatively charged 2' phosphate of NADPH, thus enabling the enzyme to utilise both NADH and NADPH. Kinetic analysis of DHDPR from *M. tuberculosis* also shows that the enzyme exhibits only a moderate preference for NADH. The crystal structures of *M. tuberculosis* DHDPR in two ternary complexes (DHDPR-2,6-PDC-NADH and DHDPR-2,6-PDC-NADPH) demonstrate that the number of hydrogen bonds between DHDPR and the nucleotides NADH and NADPH are very similar (Cirilli et al., 2003; Reddy et al., 1996; Scapin et al., 1997).
