Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H2O2. The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediates common to natural heme-containing enzymes.
Sperm glyceraldehyde-3-phosphate dehydrogenase has been shown to be a successful target for a non-hormonal contraceptive approach, but the agents tested to date have had unacceptable side effects. Obtaining the structure of the sperm-specific isoform to allow rational inhibitor design has therefore been a goal for a number of years but has proved intractable because of the insoluble nature of both native and recombinant protein. We have obtained soluble recombinant sperm glyceraldehyde-3-phosphate dehydrogenase as a heterotetramer with the Escherichia coli glyceraldehyde-3-phosphate dehydrogenase in a ratio of 1:3 and have solved the structure of the heterotetramer which we believe represents a novel strategy for structure determination of an insoluble protein.A structure was also obtained where glyceraldehyde 3-phosphate binds in the P s pocket in the active site of the sperm enzyme subunit in the presence of NAD. Modeling and comparison of the structures of human somatic and sperm-specific glyceraldehyde-3-phosphate dehydrogenase revealed few differences at the active site and hence rebut the long presumed structural specificity of 3-chlorolactaldehyde for the sperm isoform. The contraceptive activity of ␣-chlorohydrin and its apparent specificity for the sperm isoform in vivo are likely to be due to differences in metabolism to 3-chlorolactaldehyde in spermatozoa and somatic cells. However, further detailed analysis of the sperm glyceraldehyde-3-phosphate dehydrogenase structure revealed sites in the enzyme that do show significant difference compared with published somatic glyceraldehyde-3-phosphate dehydrogenase structures that could be exploited by structure-based drug design to identify leads for novel male contraceptives.Glyceraldehyde-3-phosphate dehydrogenase-S (GAPDS 3 in rat; GAPDH2 in human) is the sperm-specific isoform of GAPDH (1-3) and the sole GAPDH enzyme in sperm. GAPDS is highly conserved between species showing 94% identity between rat and mouse and 87% identity between rat and human. Within a particular species, GAPDS also shows significant sequence identity to its GAPDH paralogue, 70, 70, and 68% for rat, mouse, and human, respectively. The most striking difference between GAPDS and GAPDH is the presence of an N-terminal polyproline region in GAPDS, which is 97 residues in rat (accession number AJ297631), 105 in mouse (3), and 72 in human (2). GAPDS is restricted to the principal piece of the sperm flagellum (1, 2, 4) where it is localized to the fibrous sheath (5), an association proposed to be mediated via the N-terminal polyproline extension.GAPDS first came to prominence as a contraceptive target during the 1970s (6 -8). Investigations showed that treatment of sperm with ␣-chlorohydrin or a number of related compounds could inhibit GAPDS activity (9 -11), sperm motility (9 -13), and the fertilization of oocytes in vitro (14). The metabolite of these compounds, 3-chlorolactaldehyde (15-17), selectively inhibited GAPDS, having no effect on the activity of somatic cell GAPDH (18,19), pro...
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.
Edited by Miguel De la Rosa Keyword:Assembly mechanism Folding mechanism Formate dehydrogenase Candida methylica a b s t r a c tThe folding mechanism and stability of dimeric formate dehydrogenase from Candida methylica was analysed by exposure to denaturing agents and to heat. Equilibrium denaturation data yielded a dissociation constant of about 10 À13 M for assembly of the protein from unfolded chains and the kinetics of refolding and unfolding revealed that the overall process comprises two steps. In the first step a marginally stable folded monomeric state is formed at a rate (k 1 ) of about 2 Â 10 À3 s À1 (by deduction k À1 is about10 À4 s À1 ) and assembles into the active dimeric state with a bimolecular rate con-. The rate of dissociation of the dimeric state in physiological conditions is extremely slow (k À2 $ 3 Â 10 À7 s À1 ).
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