Structure and kinetic characterization of human sperm-specific glyceraldehyde-3-phosphate dehydrogenase, GAPDS

Abstract

hGAPDS (human sperm-specific glyceraldehyde-3-phosphate dehydrogenase) is a glycolytic enzyme essential for the survival of spermatozoa, and constitutes a potential target for non-hormonal contraception. However, enzyme characterization of GAPDS has been hampered by the difficulty in producing soluble recombinant protein. In the present study, we have overexpressed in Escherichia coli a highly soluble form of hGAPDS truncated at the N-terminus (hGAPDSΔN), and crystallized the homotetrameric enzyme in two ligand complexes. The hGAPDSΔN-NAD+-phosphate structure maps the two anion-recognition sites within the catalytic pocket that correspond to the conserved Ps site and the newly recognized Pi site identified in other organisms. The hGAPDSΔN-NAD+-glycerol structure shows serendipitous binding of glycerol at the Ps and new Pi sites, demonstrating the propensity of these anion-recognition sites to bind non-physiologically relevant ligands. A comparison of kinetic profiles between hGAPDSΔN and its somatic equivalent reveals a 3-fold increase in catalytic efficiency for hGAPDSΔN. This may be attributable to subtle amino acid substitutions peripheral to the active centre that influence the charge properties and protonation states of catalytic residues. Our data therefore elucidate structural and kinetic features of hGAPDS that might provide insightful information towards inhibitor development.

A cryoprotectant induces conformational change in glyceraldehyde-3-phosphate dehydrogenase

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The sweet side ofRNAregulation: glyceraldehyde‐3‐phosphate dehydrogenase as a noncanonicalRNA‐binding protein

“…The structures of GAPDH from archaea, bacteria, and eukaryotes have been determined by x‐ray crystallography, and show that the NAD + cofactor is bound, despite not being added during protein purification. In mammalian GAPDH structures, two, three, or even four NAD + molecules have been observed per homotetramer. In addition, we recently showed that GAPDH was present as a mixture of NAD + ‐bound and NAD + ‐free species in solution by nano‐ESI/MS/MS studies .…”

Novel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from Escherichia coli Provide New Insights into the Mechanism of Generation of 1,3-Bisphosphoglyceric Acid

“…Many structures of GAPDH from eukaryotes [ 5 , 6 , 7 ], bacteria [ 8 , 9 ], and archaea [ 10 , 11 ] have been reported. Eukaryotic and bacterial structures may have the same catalytic process, which is different from that of archaea.…”