Purification and biochemical properties of soluble recombinant human Bax.

Bax is a member of the Bcl-2 protein family with proapoptotic properties. The proteins of this family contain three highly conserved regions termed BH1, BH2, and BH3 as well as a hydrophobic COOH-terminal domain, which is responsible for the membrane attachment of the proteins. We have expressed human Bax truncated of the 20 amino acid COOH-terminal hydrophobic domain to obtain large amounts of soluble protein suitable for biochemical and structural studies. The truncated protein was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli. The GST-Bax fusion protein was bound to glutathione-Sepharose, and Bax was released by thrombin cleavage and further purified by sequential chromatography on heparin-Sepharose and DEAE-Sepharose. The purified protein was present in solution as a heptamer and multimers of the heptamer complex. Limited tryptic digestion cleaved the protein in the region preceding the BH3 domain and produced a specific stable protein fragment of 15 kDa. Phosphorylation has been proposed as a possible regulatory mechanism of the bcl-2 proteins. The Bax protein was an in vitro substrate for specific serine/threonine protein kinases.

Evidence for a double-helical structure for modular polyketide synthases.

Modular polyketide synthases are multienzymes responsible for the biosynthesis of a large number of clinically important natural products. They contain multiple sets, or modules, of enzymatic activities, distributed between a few giant multienzymes and there is one module for every successive cycle of polyketide chain extension. We show here that each multienzyme in a typical modular polyketide synthase forms a (possibly helical) parallel dimer, and that each pair of identical modules interacts closely across the dimer interface. Such an arrangement would allow identical modules to share active sites for chain extension, and thus to function independently of flanking modules, which would have important implications both for mechanisms of evolution of polyketide synthases and for their future genetic engineering.

Single amino acid substitutions disrupt tetramer formation in the dihydroneopterin aldolase enzyme of Pneumocystis carinii.

In the opportunistic pathogen Pneumocystis carinii, dihydroneopterin aldolase function is expressed as the N-terminal portion of the multifunctional folic acid synthesis protein (Fas). This region encompasses two domains, FasA and FasB, which are 27% amino acid identical. FasA and FasB also share significant amino acid sequence similarity with bacterial dihydroneopterin aldolases. In the present study, this enzyme function has been overproduced as an independent monofunctional activity in Escherichia coli. Recombinant FasAB-Met23 (amino acids 23-290 of the predicted open reading frame) was purified and shown to contain dihydroneopterin aldolase activity. The native FasAB-Met23 is a tetramer of the 30-kDa subunit, demonstrating characteristics of an associating-dissociating equilibrium system in which only the multimeric form of the enzyme is active. Multiple sequence alignment of FasA and FasB with other dihydroneopterin aldolases highlights only three positions where the amino acid is invariable between all the predicted proteins. The role of these conserved amino acid residues in enzyme function was investigated using site-directed mutagenesis. Mutant FasAB-Met23 species were overproduced and purified to near homogeneity. Three FasA domain mutants and two FasB domain mutants had little or no detectable dihydroneopterin aldolase activity, implicating both FasA and FasB in the catalytic mechanism. We show that each mutant protein containing an inactivating amino acid substitution has lost its ability to form stable tetramers.

Kinetic properties of human dopamine sulfotransferase (SULT1A3) expressed in prokaryotic and eukaryotic systems: comparison with the recombinant enzyme purified from Escherichia coli.

Sulfation, catalyzed by members of the sulfotransferase enzyme family, is a major metabolic pathway which modulates the biological activity of numerous endogenous and xenobiotic chemicals. A number of these enzymes have been expressed in prokaryotic and eukaryotic systems to produce protein for biochemical and physical characterization. However, the effective use of heterologous expression systems to produce recombinant enzymes for such purposes depends upon the expressed protein faithfully representing the "native" protein. For human sulfotransferases, little attention has been paid to this despite the widespread use of recombinant enzymes. Here we have validated a number of heterologous expression systems for producing the human dopamine-metabolizing sulfotransferase SULT1A3, including Escherichia coli, Saccharomyces cerevisiae, COS-7, and V79 cells, by comparison of Km values of the recombinant enzyme in cell extracts with enzyme present in human platelets and with recombinant enzyme purified to homogeneity following E. coli expression. This is the first report of heterologous expression of a cytosolic sulfotransferase in yeast. Expression of SULT1A3 was achieved in all cell types, and the Km for dopamine under the conditions applied was approximately 1 microM in all heterologous systems studied, which compared favorably with the value determined with human platelets. We also determined the subunit and native molecular weights of the purified recombinant enzyme by SDS-PAGE, electrospray ionization mass spectrometry, dynamic light scattering, and sedimentation analysis. The enzyme purified following expression in E. coli existed as a homodimer with Mr approximately 68,000 as determined by light scattering and sedimentation analysis. Mass spectrometry revealed two species with experimentally determined masses of 34,272 and 34,348 which correspond to the native protein with either one or two 2-mercaptoethanol adducts. We conclude that the enzyme expressed in prokaryotic and eukaryotic heterologous systems, and also purified from E. coli, equates to that which is found in human tissue preparations.

Novel natural product 5,5-trans-lactone inhibitors of human alpha-thrombin: mechanism of action and structural studies.

High-throughput screening of methanolic extracts from the leaves of the plant Lantana camara identified potent inhibitors of human alpha-thrombin, which were shown to be 5,5-trans-fused cyclic lactone euphane triterpenes [O'Neill et al. (1998) J. Nat. Prod. (submitted for publication)]. Proflavin displacement studies showed the inhibitors to bind at the active site of alpha-thrombin and alpha-chymotrypsin. Kinetic analysis of alpha-thrombin showed tight-binding reversible competitive inhibition by both compounds, named GR133487 and GR133686, with respective kon values at pH 8.4 of 1.7 x 10(6) s-1 M-1 and 4.6 x 10(6) s-1 M-1. Electrospray ionization mass spectrometry of thrombin/inhibitor complexes showed the tight-bound species to be covalently attached, suggesting acyl-enzyme formation by reaction of the active-site Ser195 with the trans-lactone carbonyl. X-ray crystal structures of alpha-thrombin/GR133686 (3.0 A resolution) and alpha-thrombin/GR133487 (2.2 A resolution) complexes showed continuous electron density between Ser195 and the ring-opened lactone carbonyl, demonstrating acyl-enzyme formation. Turnover of inhibitor by alpha-thrombin was negligible and mass spectrometry of isolated complexes showed that reversal of inhibition occurs by reformation of the trans-lactone from the acyl-enzyme. The catalytic triad appears undisrupted and the inhibitor carbonyl occupies the oxyanion hole, suggesting the observed lack of turnover is due to exclusion of water for deacylation. The acyl-enzyme inhibitor hydroxyl is properly positioned for nucleophilic attack on the ester carbonyl and therefore relactonization; furthermore, the higher resolution structure of alpha-thrombin/GR133487 shows this hydroxyl to be effectively superimposable with the recently proposed deacylating water for peptide substrate hydrolysis [Wilmouth, R. C., et al. (1997) Nat. Struct.Biol. 4, 456-462], suggesting the alpha-thrombin/GR133487 complex may be a good model for this reaction.