The availability of published genomes from all domains of life has provided insight into biochemical processes for many organisms. Frequently the mapping of classical pathways onto genome-derived data is used to deduce metabolic pathways in an otherwise uncharacterised system. Whilst this method may be sufficient as a prelude to further biochemical analysis, the function of genes may be assigned by extrapolation from homologs, and this may not be correct. This study highlights the dangers of such a process, focusing on the glycolytic/gluconeogenic enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from the human pathogenic species Helicobacter pylori and Campylobacter jejuni. H. pylori has two genes encoding GAPDH (gapA and gapB). These are both annotated as NAD+- dependent glyceraldehyde 3-phosphate dehydrogenases. This study has demonstrated enzymatically and structurally that gapA encodes a NADP-dependent GAPDH, whilst gapB encodes a NAD+-dependent GAPDH, furthermore GAPDHB is a better phosphorylating erythrose-4-phosphate dehydrogenase. Structural analysis of GAPDHA and GAPDHB showed key residues providing specificity for the coenzyme NADP+ over NAD+ and this finding was used to search for other putative NADP+-dependent GAPDHs within the Campylobacterales order. Other NADP+-dependent GAPDHs were identified; including that of C. jejuni, which has only one annotated GAPDH-encoding gene. Structural and enzymatic analysis confirmed C. jejuni's GAPDH is NADP+ dependent, although dual specificity is observed. This further shows the importance of experimental data to describe a system.\ud Finally, a mutagenic approach was undertaken to determine the mechanism underlying the differing substrate specificities between GAPDHA and GAPDHB. Whilst the structural analysis was unable to provide a determinant of substrate specificity, these structures provided clear evidence for a reaction mechanism used by all phosphorylating GAPDHs. The significance of the findings is discussed in the context of the metabolism of these pathogens.\ud This work demonstrates the importance of the synergy between structural and genomic analysis
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