Crystal structure of the 6-phosphogluconate dehydrogenase from Gluconobacter oxydans reveals tetrameric 6PGDHs as the crucial intermediate in the evolution of structure and cofactor preference in the 6PGDH family
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Media type:
E-Article
Title:
Crystal structure of the 6-phosphogluconate dehydrogenase from Gluconobacter oxydans reveals tetrameric 6PGDHs as the crucial intermediate in the evolution of structure and cofactor preference in the 6PGDH family
Description:
<ns4:p><ns4:bold>Background: </ns4:bold>The enzyme 6-phosphogluconate dehydrogenase (6PGDH) is the central enzyme of the oxidative pentose phosphate pathway. Members of the 6PGDH family belong to different classes: either homodimeric enzymes assembled from long-chain subunits or homotetrameric ones assembled from short-chain subunits. Dimeric 6PGDHs bear an internal duplication absent in tetrameric 6PGDHs and distant homologues of the β-hydroxyacid dehydrogenase (βHADH) superfamily.</ns4:p><ns4:p> <ns4:bold>Methods: </ns4:bold>We use X-ray crystallography to determine the structure of the apo form of the 6PGDH from <ns4:italic>Gluconobacter oxydans </ns4:italic>(<ns4:italic>Go</ns4:italic>6PGDH). We carried out a structural and phylogenetic analysis of short and long-chain 6PGDHs. We put forward an evolutionary hypothesis explaining the differences seen in oligomeric state vs. dinucleotide preference of the 6PGDH family. We determined the cofactor preference of <ns4:italic>Go</ns4:italic>6PGDH at different 6-phosphogluconate concentrations, characterizing the wild-type enzyme and three-point mutants of residues in the cofactor binding site of <ns4:italic>Go</ns4:italic>6PGDH.</ns4:p><ns4:p> <ns4:bold>Results: </ns4:bold>The structural comparison suggests that the 6PG binding site initially evolved by exchanging C-terminal α-helices between subunits. An internal duplication event changed the quaternary structure of the enzyme from a tetrameric to a dimeric arrangement. The phylogenetic analysis suggests that 6PGDHs have spread from Bacteria to Archaea and Eukarya on multiple occasions by lateral gene transfer. Sequence motifs consistent with NAD<ns4:sup>+</ns4:sup>- and NADP<ns4:sup>+</ns4:sup>-specificity are found in the β2-α2 loop of dimeric and tetrameric 6PGDHs. Site-directed mutagenesis of <ns4:italic>Go</ns4:italic>6PGDH inspired by this analysis fully reverses dinucleotide preference. One of the mutants we engineered has the highest efficiency and specificity for NAD<ns4:sup>+</ns4:sup> so far described for a 6PGDH.</ns4:p><ns4:p> <ns4:bold>Conclusions: </ns4:bold>The family 6PGDH comprises dimeric and tetrameric members whose active sites are conformed by a C-terminal α-helix contributed from adjacent subunits. Dimeric 6PGDHs have evolved from the duplication-fusion of the tetrameric C-terminal domain before independent transitions of cofactor specificity. Changes in the conserved β2-α2 loop are crucial to modulate the cofactor specificity in <ns4:italic>Go</ns4:italic>6PGDH.</ns4:p>