ABSTRACT
The crystallization of 16 proteins was carried out using 60 wells on board Shenzhou 3 in 2002. Although the mission was only 7 days, careful and concerted planning at all stages made it possible to obtain crystals of improved quality compared to their ground controls for some of the proteins. Significantly improved resolutions were obtained from diffracted crystals of 4 proteins. A complete data set from a space crystal of the PEP carboxykinase yielded significantly higher resolution (1.46A vs. 1.87A), I/sigma (22.4 vs. 15.5), and a lower average temperature factor (29.2A(2) vs. 42.9A(2)) than the best ground-based control crystal. The 3-D structure of the enzyme is well improved with significant ligand density. It has been postulated that the reduced convection and absence of macromolecule sedimentation under microgravity have advantages/benefits for protein crystal growth. Improvements in experimental design for protein crystal growth in microgravity are ongoing.
Subject(s)
Crystallization/methods , Crystallography, X-Ray/methods , Cytochromes b5/chemistry , Electrons , Escherichia coli/enzymology , Fourier Analysis , Humans , Models, Molecular , Phosphoenolpyruvate Carboxykinase (ATP)/chemistry , Signal Transduction , Software , Space Flight , Temperature , WeightlessnessABSTRACT
The first crystallographic structure of human type 3 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD3, AKR1C2), an enzyme playing a critical role in steroid hormone metabolism, has been determined in complex with testosterone and NADP at 1.25-A resolution. The enzyme's 17beta-HSD activity was studied in comparison with its 3alpha-HSD activity. The enzyme catalyzes the inactivation of dihydrotestosterone into 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) as well as the transformation of androstenedione into testosterone. Using our homogeneous and highly active enzyme preparation, we have obtained 150-fold higher 3alpha-HSD specificity as compared with the former reports in the literature. Although the rat and the human 3alpha-HSDs share 81% sequence homology, our structure reveals significantly different geometries of the active sites. Substitution of the Ser(222) by a histidine in the human enzyme may compel the steroid to adopt a different binding to that previously described for the rat (Bennett, M. J., Albert, R. H., Jez, J. M., Ma, H., Penning, T. M., and Lewis, M. (1997) Structure 5, 799-T812). Furthermore, we showed that the affinity for the cofactor is higher in the human 3alpha-HSD3 than the rat enzyme due to the presence of additional hydrogen bonds on the adenine moiety and that the cofactor is present under its reduced form in the active site in our preparation.
Subject(s)
3-Hydroxysteroid Dehydrogenases/chemistry , Isoenzymes/chemistry , NADP/chemistry , Testosterone/chemistry , 17-Hydroxysteroid Dehydrogenases/metabolism , 3-Hydroxysteroid Dehydrogenases/metabolism , 3-alpha-Hydroxysteroid Dehydrogenase (B-Specific) , Binding Sites , HumansABSTRACT
Mammalian alpha-amylases catalyze the hydrolysis of alpha-linked glucose polymers according to a complex processive mechanism. We have determined the X-ray structures of porcine pancreatic alpha-amylase complexes with the smallest molecule of the trestatin family (acarviosine-glucose) which inhibits porcine pancreatic alpha-amylase and yet is not hydrolyzed by the enzyme. A structure analysis at 1.38 A resolution of this complex allowed for a clear identification of a genuine single hexasaccharide species composed of two alpha-1,4-linked original molecules bound to the active site of the enzyme. The structural results supported by mass spectrometry experiments provide evidence for an enzymatically catalyzed condensation reaction in the crystal.
Subject(s)
Amino Sugars/chemistry , Amino Sugars/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , alpha-Amylases/antagonists & inhibitors , alpha-Amylases/chemistry , Acarbose/metabolism , Animals , Anions , Binding Sites , Catalysis , Chlorides/metabolism , Cold Temperature , Computer Simulation , Disulfides/chemistry , Humans , Hydrolysis , Ligands , Models, Molecular , Pancreas/enzymology , Protein Conformation , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Swine , alpha-Amylases/metabolismABSTRACT
In androgen-sensitive target tissues, 3 alpha-hydroxysteroid dehydrogenase regulates the androgen receptor (AR) activity by catalyzing the inactivation of 5 alpha-dihydrotestosterone (the most natural potent androgen) to 5 alpha-androstane-3 alpha,17 beta-diol. In this report, the crystallization of a human prostatic type 3 3 alpha-hydroxysteroid dehydrogenase, a member of the aldo-keto reductase superfamily, is described. Two different crystal forms of the complex between the human type 3 3 alpha-HSD, NADP(+) and testosterone have been obtained using PEG as precipitant. Crystal form I, which diffracts to 1.6 A, belongs to the monoclinic space group P2(1), with unit-cell parameters a = 55.07, b = 87.15, c = 76.88 A, beta = 107.37 degrees and two subunits in the asymmetric unit. A complete data set has been collected at 1.8 A. Crystal form II, which diffracts to 2.6 A, belongs to the rhombohedral space group R32, with unit-cell parameters a = b = 143.59, c = 205.86 A, alpha = beta = 90, gamma = 120 degrees and two subunits in the asymmetric unit.
Subject(s)
3-Hydroxysteroid Dehydrogenases/chemistry , Prostate/enzymology , 3-Hydroxysteroid Dehydrogenases/classification , 3-Hydroxysteroid Dehydrogenases/metabolism , Binding Sites , Crystallization , Humans , Male , Protein Subunits , Substrate Specificity , Testosterone/metabolism , X-Ray DiffractionABSTRACT
Crystal structures of human pancreatic alpha-amylase (HPA) in complex with naturally occurring inhibitors have been solved. The tetrasaccharide acarbose and a pseudo-pentasaccharide of the trestatin family produced identical continuous electron densities corresponding to a pentasaccharide species, spanning the -3 to +2 subsites of the enzyme, presumably resulting from transglycosylation. Binding of the acarviosine core linked to a glucose residue at subsites -1 to +2 appears to be a critical part of the interaction process between alpha-amylases and trestatin-derived inhibitors. Two crystal forms, obtained at different values of pH, for the complex of HPA with the protein inhibitor from Phaseolus vulgaris (alpha-amylase inhibitor) have been solved. The flexible loop typical of the mammalian alpha-amylases was shown to exist in two different conformations, suggesting that loop closure is pH-sensitive. Structural information is provided for the important inhibitor residue, Arg-74, which has not been observed previously in structural analyses.
Subject(s)
Enzyme Inhibitors/metabolism , Lectins/metabolism , Pancreas/enzymology , Plant Lectins , Plant Proteins/metabolism , Polysaccharides/metabolism , alpha-Amylases/chemistry , alpha-Amylases/metabolism , Acarbose/chemistry , Acarbose/metabolism , Acarbose/pharmacology , Amino Sugars/metabolism , Amino Sugars/pharmacology , Arginine/chemistry , Arginine/metabolism , Binding Sites , Crystallography, X-Ray , Electrons , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Glycosylation , Humans , Hydrogen Bonding , Hydrogen-Ion Concentration , Lectins/chemistry , Lectins/pharmacology , Models, Molecular , Molecular Sequence Data , Plant Proteins/chemistry , Plant Proteins/pharmacology , Polysaccharides/chemistry , Polysaccharides/pharmacology , Protein Conformation , Trisaccharides/chemistry , Trisaccharides/metabolism , Trisaccharides/pharmacology , alpha-Amylases/antagonists & inhibitorsABSTRACT
The alpha-amylase from Tenebrio molitor larvae (TMA) has been crystallized in complex with the alpha-amylase inhibitor (alpha-AI) from the bean Phaseolus vulgaris. A molecular-replacement solution of the structure was obtained using the refined pig pancreatic alpha-amylase (PPA) and alpha-AI atomic coordinates as starting models. The structural analysis showed that although TMA has the typical structure common to alpha-amylases, large deviations from the mammalian alpha-amylase models occur in the loops. Despite these differences in the interacting loops, the bean inhibitor is still able to inhibit both the insect and mammalian alpha-amylase.
