Structural basis for inhibition of 17ß-hydroxysteroid dehydrogenases by phytoestrogens: The case of fungal 17ß-HSDcl.

Phytoestrogens are plant-derived compounds that functionally and structurally mimic mammalian estrogens. Phytoestrogens have broad inhibitory activities toward several steroidogenic enzymes, such as the 17ß-hydroxysteroid dehydrogenases (17ß-HSDs), which modulate the biological potency of androgens and estrogens in mammals. However, to date, no crystallographic data are available to explain phytoestrogens binding to mammalian 17ß-HSDs. NADP(H)-dependent 17ß-HSD from the filamentous fungus Cochliobolus lunatus (17ß-HSDcl) has been the subject of extensive biochemical, kinetic and quantitative structure-activity relationship studies that have shown that the flavonols are the most potent inhibitors. In the present study, we investigated the structure-activity relationships of the ternary complexes between the holo form of 17ß-HSDcl and the flavonols kaempferol and 3,7-dihydroxyflavone, in comparison with the isoflavones genistein and biochanin A. Crystallographic data are accompanied by kinetic analysis of the inhibition mechanisms for six flavonols (3-hydroxyflavone, 3,7-dihydroxyflavone, kaempferol, quercetin, fisetin, myricetin), one flavanone (naringenin), one flavone (luteolin), and two isoflavones (genistein, biochanin A). The kinetics analysis shows that the degree of hydroxylation of ring B significantly influences the overall inhibitory efficacy of the flavonols. A distinct binding mode defines the interactions between 17ß-HSDcl and the flavones and isoflavones. Moreover, the complex with biochanin A reveals an unusual binding mode that appears to account for its greater inhibition of 17ß-HSDcl with respect to genistein. Overall, these data provide a blueprint for identification of the distinct molecular determinants that underpin 17ß-HSD inhibition by phytoestrogens.

Second-sphere interactions between the C93-Y157 cross-link and the substrate-bound Fe site influence the O2 coupling efficiency in mouse cysteine dioxygenase.

Cysteine dioxygenase (CDO) is a non-heme iron enzyme that catalyzes the O2-dependent oxidation of l-cysteine (l-Cys) to produce cysteinesulfinic acid (CSA). Adjacent to the Fe site of CDO is a covalently cross-linked cysteine-tyrosine pair (C93-Y157). While several theories have been proposed for the function of the C93-Y157 pair, the role of this post-translational modification remains unclear. In this work, the steady-state kinetics and O2/CSA coupling efficiency were measured for wild-type CDO and selected active site variants (Y157F, C93A, and H155A) to probe the influence of second-sphere enzyme-substrate interactions on catalysis. In these experiments, it was observed that both kcat and the O2/CSA coupling efficiency were highly sensitive to the presence of the C93-Y157 cross-link and its proximity to the substrate carboxylate group. Complementary electron paramagnetic resonance (EPR) experiments were performed to obtain a more detailed understanding of the second-sphere interactions identified in O2/CSA coupling experiments. Samples of the catalytically inactive substrate-bound Fe(III)-CDO species were treated with cyanide, resulting in a low-spin (S = ¹/2) ternary complex. Remarkably, both the presence of the C93-Y157 pair and interactions with the Cys carboxylate group could be readily identified by perturbations to the rhombic EPR signal. Spectroscopically validated active site quantum mechanics/molecular mechanics and density functional theory computational models are provided to suggest a potential role for Y157 in the positioning of the substrate Cys in the active site and to verify the orientation of the g-tensor relative to the CDO Fe site molecular axis.

Characterization of methylmalonyl-CoA mutase involved in the propionate photoassimilation of Euglena gracilis Z.

Significant accumulation of the methylmalonyl-CoA mutase apoenzyme was observed in the photosynthetic flagellate Euglena gracilis Z at the end of the logarithmic growth phase. The apoenzyme was converted to a holoenzyme by incubation for 4 h at 4 degrees C with 10 microM 5'-deoxyadenosylcobalamin, and then, the holoenzyme was purified to homogeneity and characterized. The apparent molecular mass of the enzyme was calculated to be 149.0 kDa +/- 5.0 kDa using Superdex 200 gel filtration. SDS-polyacrylamide gel electrophoresis of the purified enzyme yielded a single protein band with an apparent molecular mass of 75.0 kDa +/- 3.0 kDa, indicating that the Euglena enzyme is composed of two identical subunits. The purified enzyme contained one mole of prosthetic 5'-deoxyadenosylcobalamin per mole of the enzyme subunit. Moreover, we cloned the full-length cDNA of the Euglena enzyme. The cDNA clone contained an open reading frame encoding a protein of 717 amino acids with a calculated molecular mass of 78.3 kDa, preceded by a putative mitochondrial targeting signal consisting of nine amino acid residues. Furthermore, we studied some properties and physiological function of the Euglena enzyme.

Functional maps of the junctions between interglobular contacts and active sites in glycolytic enzymes -- a comparative analysis of the biochemical and structural data.

BACKGROUND: Oligomers and separate subunits of the glycolytic enzymes often have different catalytic properties. However, spectral data show an apparent lack of significant conformational changes during oligomerization. Since the conformation of an enzyme determines its catalytic properties, the structural mechanism(s) influencing the activity is of considerable interest. MATERIAL/METHODS: Analysis of the spatial structures of the junctions between interglobular contacts and binding sites may give a clue to the mechanism(s) of the activation. In this work, the problem was studied using available structural and biochemical data for the oligomeric enzymes of glycolysis. RESULTS: Computational analysis of the structures of the junctions has identified three structurally distinct types of junctions: 1. interglobular binding site (2 of 8 enzymes); 2. domain-domain stabilization (5 of 8); and 3. 'sequence overlap' or a local conformational change (all enzymes). Thus the catalytic activity may be influenced through the shifts of the modules of protein structure (types 1, 2) and/or due to a slight change in the local structure (type 3). The more common junctions of types 2 and 3 are well conserved among eukaryotic enzymes, which suggests their biological importance. CONCLUSIONS: The results suggest that a profound and a complex change in conformation in subunits of an oligomeric enzyme may not be necessary for a significant change in the catalytic properties. The analysis maps the residues important for the junctions and thus for the link between the catalytic activity and the oligomeric state of the enzymes.

Catalytic mechanism of S-adenosylhomocysteine hydrolase. Site-directed mutagenesis of Asp-130, Lys-185, Asp-189, and Asn-190.

S-Adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of S-adenosylhomocysteine to form adenosine and homocysteine. On the bases of crystal structures of the wild type enzyme and the D244E mutated enzyme complexed with 3'-keto-adenosine (D244E.Ado*), we have identified the important amino acid residues, Asp-130, Lys-185, Asp-189, and Asn-190, for the catalytic reaction and have proposed a catalytic mechanism (Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (2000) J. Biol. Chem. 275, 32147-32156). To confirm the proposed catalytic mechanism, we have made the D130N, K185N, D189N, and N190S mutated enzymes and measured the catalytic activities. The catalytic rates (k(cat)) of D130N, K185N, D189N, and N190S mutated enzymes are reduced to 0.7%, 0.5%, 0.1%, and 0.5%, respectively, in comparison with the wild type enzyme, indicating that Asp-130, Lys-185, Asp-189, and Asn-190 are involved in the catalytic reaction. K(m) values of the mutated enzymes are increased significantly, except for the N190S mutation, suggesting that Asp-130, Lys-185, and Asp-189 participate in the substrate binding. To interpret the kinetic data, the oxidation states of the bound NAD molecules of the wild type and mutated enzymes were measured during the catalytic reaction by monitoring the absorbance at 340 nm. The crystal structures of the WT and D244E.Ado*, containing four subunits in the crystallographic asymmetric unit, were re-refined to have the same subunit structures. A detailed catalytic mechanism of AdoHcyase has been revealed based on the oxidation states of the bound NAD and the re-refined crystal structures of WT and D244E.Ado*. Lys-185 and Asp-130 abstract hydrogen atoms from 3'-OH and 4'-CH, respectively. Asp-189 removes a proton from Lys-185 and produces the neutral N zeta (-NH(2)), and Asn-190 facilitates formation of the neutral Lys-185. His-54 and His-300 hold and polarize a water molecule, which nucleophilically attacks the C5'- of 3'-keto-4',5'-dehydroadenosine to produce 3'-keto-Ado.

Unconventional mRNA processing in the expression of two calcineurin B isoforms in Dictyostelium.

The genome of Dictyostelium discoideum contains a single gene (cnbA) for the regulatory (B) subunit of the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin (CN). Two mRNA species and two protein products differing in size were found. The apparent molecular masses of the protein isoforms corresponded to translation products starting from the first and second AUG codons of the primary transcript, respectively. The smaller mRNA and protein isoforms accumulated during early differentiation of the cells. Whereas the amount of the higher molecular mass protein isoform remained constant throughout development, the larger mRNA disappeared to virtually undetectable levels during aggregation. 5'RACE amplification of the smaller transcript yielded cDNAs lacking the 5' non-translated region and the first ATG initiator codon. Expression of truncated cDNAs and various chimeric genes encoding CNB-green fluorescent protein fusions in Dictyostelium indicate that the mature cnbA transcript is processed by an unconventional mechanism that leads to truncation of the 5' untranslated region and at least the first AUG initiator codon, and to utilization of the second AUG codon for translation initiation of the small CNB isoform. Determinants for this processing mechanism reside within the coding region of the cnbA gene.

Structural analysis of glyceraldehyde 3-phosphate dehydrogenase from Escherichia coli: direct evidence of substrate binding and cofactor-induced conformational changes.

The crystal structures of gyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Escherichia coli have been determined in three different enzymatic states, NAD(+)-free, NAD(+)-bound, and hemiacetal intermediate. The NAD(+)-free structure reported here has been determined from monoclinic and tetragonal crystal forms. The conformational changes in GAPDH induced by cofactor binding are limited to the residues that bind the adenine moiety of NAD(+). Glyceraldehyde 3-phosphate (GAP), the substrate of GAPDH, binds to the enzyme with its C3 phosphate in a hydrophilic pocket, called the "new P(i)" site, which is different from the originally proposed binding site for inorganic phosphate. This observed location of the C3 phosphate is consistent with the flip-flop model proposed for the enzyme mechanism [Skarzynski, T., Moody, P. C., and Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187]. Via incorporation of the new P(i) site in this model, it is now proposed that the C3 phosphate of GAP initially binds at the new P(i) site and then flips to the P(s) site before hydride transfer. A superposition of NAD(+)-bound and hemiacetal intermediate structures reveals an interaction between the hydroxyl oxygen at the hemiacetal C1 of GAP and the nicotinamide ring. This finding suggests that the cofactor NAD(+) may stabilize the transition state oxyanion of the hemiacetal intermediate in support of the flip-flop model for GAP binding.

Structure of apo-glyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor.

d-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) shows cooperative properties for binding coenzymes. The structure of apo-GAPDH from Palinurus versicolor has been solved at 2.0 A resolution by X-ray crystallography. The final model gives a crystallographic R factor of 0.178 in the resolution range 8 to 2 A. The structural comparison with holo-GAPDH from the same species reveals a conformational change induced by coenzyme binding similar to that observed in Bacillus stearothermophilus GAPDH but to a lesser extent. The differences in magnitude during the apo-holo transition between these two enzymes were analyzed with respect to the change of the amino acid composition in the coenzyme binding pocket. In the crystalline state of apo-GAPDH, the overall structures of the subunits are similar to each other; however, significant differences in temperature factors and minor differences in domain rotation upon coenzyme binding were observed for different subunits. These structural features are discussed in relation to the environmental asymmetry of crystallographically independent subunits.

Structure of active site carboxymethylated D-glyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor.

The structure of active site carboxymethylated D-glyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor was determined in the presence of coenzyme NAD+ at 1.88 A resolution with a final R-factor of 0.175. The structure refinement was carried out on the basis of the structure of holo-GAPDH at 2.0 A resolution using the program XPLOR. The carboxymethyl group connected to Cys149 is stabilized by a hydrogen bond between its OZ1 and Cys149N, and charge interaction between the carboxyl group and the nicotinamide moiety. The modification of Cys149 induced conformational changes in the active site, in particular, the site of sulphate ion 501 (the proposed attacking inorganic phosphate ion in catalysis), and segment 208-218 nearby. Extensive hydrogen-bonding interactions occur in the active site, which contribute to the higher stability of the modified enzyme. The modification of the active site did not affect the conformation of GAPDH elsewhere, including the subunit interfaces. The structures of the green and red subunits in the asymmetric unit are nearly identical, suggesting that the half-site reactivity of this enzyme is from ligand-induced rather than pre-existing asymmetry. It is proposed that the carboxymethyl group takes the place of the acyl group of the reaction intermediate, and the catalytic mechanism of this enzyme is discussed in the light of a comparison of the structures of the native and the carboxymethylated GAPDH.