A new triclinic crystal form of human serum albumin (HSA), derived either from pool plasma (pHSA) or from a Pichia pastoris expression system (rHSA), was obtained from polyethylene glycol 4000 solution. Three-dimensional structures of pHSA and rHSA were determined at 2.5 A resolution from the new triclinic crystal form by molecular replacement, using atomic coordinates derived from a multiple isomorphous replacement work with a known tetragonal crystal form. The structures of pHSA and rHSA are virtually identical, with an r.m. s. deviation of 0.24 A for all Calpha atoms. The two HSA molecules involved in the asymmetric unit are related by a strict local twofold symmetry such that the Calpha atoms of the two molecules can be superimposed with an r.m.s. deviation of 0.28 A in pHSA. Cys34 is the only cysteine with a free sulfhydryl group which does not participate in a disulfide linkage with any external ligand. Domains II and III both have a pocket formed mostly of hydrophobic and positively charged residues and in which a very wide range of compounds may be accommodated. Three tentative binding sites for long-chain fatty acids, each with different surroundings, are located at the surface of each domain.

The three-dimensional structure of D-amino acid aminotransferase (D-AAT) in the pyridoxamine phosphate form has been determined crystallographically. The fold of this pyridoxal phosphate (PLP)-containing enzyme is completely different from those of any of the other enzymes that utilize PLP as part of their mechanism and whose structures are known. However, there are some striking similarities between the active sites of D-AAT and the corresponding enzyme that transaminates L-amino acids, L-aspartate aminotransferase. These similarities represent convergent evolution to a common solution of the problem of enforcing transamination chemistry on the PLP cofactor. Implications of these similarities are discussed in terms of their possible roles in the stabilization of intermediates of a transamination reaction. In addition, sequence similarity between D-AAT and branched chain L-amino acid aminotransferase suggests that this latter enzyme will also have a fold similar to that of D-AAT.

The crystal structure of recombinant chicken triosephosphate isomerase (TIM, E.C. 5.3.1.1) complexed with the intermediate analogue phosphoglycolohydroxamate (PGH) has been solved by the method of molecular replacement and refined to an R-factor of 18.5% at 1.8-A resolution. The structure is essentially identical to that of the yeast TIM-PGH complex [Davenport, R. C., et al. (1991) Biochemistry 30, 5821-5826] determined earlier and refined at comparable resolution. This identity extends to the high-energy conformations of the active-site residues Lys13 and Ser211, as well as the positions of several bound water molecules that are retained in the active site when PGH is bound. Comparison with the structure of uncomplexed chicken TIM shows that the catalytic base, Glu165, moves several angstroms when PGH binds. This movement may provide a trigger for a larger conformational change, one of 7 A, in a loop near the active site, which folds down like a lid to shield the bound inhibitor and catalytic residues from contact with bulk solvent. These same conformational changes were seen in crystalline yeast TIM upon binding of PGH; their occurrence here in a different crystal form of TIM eliminates the possibility that they are an artifact of crystal packing.

The crystal structures of a wild-type and a mutant PCS, a novel plant type III polyketide synthase from a medicinal plant, Aloe arborescens, were solved at 1.6 A resolution. The crystal structures revealed that the pentaketide-producing wild-type and the octaketide-producing M207G mutant shared almost the same overall folding, and that the large-to-small substitution dramatically increases the volume of the polyketide-elongation tunnel by opening a gate to two hidden pockets behind the active site of the enzyme. The chemically inert active site residue 207 thus controls the number of condensations of malonyl-CoA, solely depending on the steric bulk of the side chain. These findings not only provided insight into the polyketide formation reaction, but they also suggested strategies for the engineered biosynthesis of polyketides.

The structural basis for the effect of the S96P mutation in chicken triosephosphate isomerase (cTIM) has been analyzed using a combination of X-ray crystallography and Fourier transform infrared spectroscopy. The X-ray structure is that of the enzyme complexed with phosphoglycolohydroxamate (PGH), an intermediate analogue, solved at a resolution of 1.9 A. The S96P mutation was identified as a second-site reverent when catalytically crippled mutants, E165D and H95N, were subjected to random mutagenesis. The presence of the second mutation leads to enhanced activity over the single mutation. However, the effect of the S96P mutation alone is to decrease the catalytic efficiency of the enzyme. The crystal structures of the S96P double mutants show that this bulky proline side chain alters the water structure within the active-site cavity (E165D; ref 1) and prevents nonproductive binding conformations of the substrate (H95N; ref 2). Comparison of the S96P single mutant structure with those of the wild-type cTIM, those of the single mutants (E165D and H95N), and those of the double mutants (E165D/S96P and H95N/S96P) begins to address the role of the conserved serine residue at this position. The results indicate that the residue positions the catalytic base E165 optimally for polarization of the substrate carbonyl, thereby aiding in proton abstraction. In addition, this residue is involved in positioning critical water molecules, thereby affecting the way in which water structure influences activity.

Ficolins are a kind of pathogen-recognition molecule in the innate immune systems. To investigate the discrimination mechanism between self and non-self by ficolins, we determined the crystal structure of the human M-ficolin fibrinogen-like domain (FD1), which is the ligand-binding domain, at 1.9 Å resolution. Although the FD1 monomer shares a common fold with the fibrinogen ␥ fragment and tachylectin-5A, the Asp-282-Cys-283 peptide bond, which is the predicted ligand-binding site on the C-terminal P domain, is a normal trans bond, unlike the cases of the other two proteins. The trimeric formation of FD1 results in the separation of the three P domains, and the spatial arrangement of the three predicted ligand-binding sites on the trimer is very similar to that of the trimeric collectin, indicating that such an arrangement is generally required for pathogen-recognition. The ligand binding study of FD1 in solution indicated that the recombinant protein binds to N-acetyl-D-glucosamine and the peptide Gly-Pro-Arg-Pro and suggested that the ligand-binding region exhibits a conformational equilibrium involving cis-trans isomerization of the Asp-282-Cys-283 peptide bond. The crystal structure and the ligand binding study of FD1 provide an insight of the self-and non-self discrimination mechanism by ficolins.Surveillance systems of innate immunity are present in all multicellular organisms and play a crucial role in the first line of defense against pathogens. Ficolins, as well as collectins, are one of the most important groups of pattern recognition molecules in the innate immunity systems (1-7) and have been identified in both vertebrates and invertebrates (6). Ficolins are comprised of a collagen-like domain at the N terminus and a fibrinogen-like domain (FBG), 3 which is the sugar-binding site, at the C terminus (8, 9). Collectins, such as mannose-binding lectin (MBL), lung surfactant protein A, and surfactant protein D, also consist of an N-terminal collagen-like domain and a C-terminal carbohydrate-recognition domain (CRD) that binds to certain carbohydrates such as mannose and GlcNAc Ca 2ϩ dependently. The CRD on MBL, surfactant protein A, and surfactant protein D forms a trimeric structure through a triple ␣-helical coiled-coil at a short neck region between the collagen-like domain and the CRD (10 -12). Ficolins also form trimers (8,13,14), although the mechanism of trimerization is unclear. Both ficolins and collectins form trimer-based multimers that are N-terminally linked by disulfide bonds (15). Ficolins and MBL also interact with MBL-associated serine proteases, and their complexes activate the lectin complement pathway (6, 16 -23).Ficolins were originally discovered in porcine uterus membrane extracts as transforming growth factor-␤-binding proteins (24,25). In human, L-ficolin and H-ficolin in serum and M-ficolin in cells have been characterized (9, 14, 26 -29). Lficolin (synonymous with ficolin-2 or Ficolin/P35) binds to GlcNAc (29, 30) and GalNAc (8). The binding ability is inhibited by acetylated c...

Curcuminoid synthase (CUS) from Oryza sativa is a plant-specific type III polyketide synthase (PKS) that catalyzes the remarkable one-pot formation of the C 6 -C 7 -C 6 diarylheptanoid scaffold of bisdemethoxycurcumin, by the condensation of two molecules of 4-coumaroyl-CoA and one molecule of malonyl-CoA. The crystal structure of O. sativa CUS was solved at 2.5-Å resolution, which revealed a unique, downward expanding active-site architecture, previously unidentified in the known type III PKSs. The large active-site cavity is long enough to accommodate the two C 6 -C 3 coumaroyl units and one malonyl unit. Furthermore, the crystal structure indicated the presence of a putative nucleophilic water molecule, which forms hydrogen bond networks with Ser351-Asn142-H 2 O-Tyr207-Glu202, neighboring the catalytic Cys174 at the active-site center. These observations suggest that CUS employs unique catalytic machinery for the one-pot formation of the C 6 -C 7 -C 6 scaffold. Thus, CUS utilizes the nucleophilic water to terminate the initial polyketide chain elongation at the diketide stage. Thioester bond cleavage of the enzyme-bound intermediate generates 4-coumaroyldiketide acid, which is then kept within the downward expanding pocket for subsequent decarboxylative condensation with the second 4-coumaroyl-CoA starter, to produce bisdemethoxycurcumin. The structure-based site-directed mutants, M265L and G274F, altered the substrate and product specificities to accept 4-hydroxyphenylpropionyl-CoA as the starter to produce tetrahydrobisdemethoxycurcumin. These findings not only provide a structural basis for the catalytic machinery of CUS but also suggest further strategies toward expanding the biosynthetic repertoire of the type III PKS enzymes.

Benzalacetone synthase (BAS), a plant-specific type III polyketide synthase (PKS), catalyzes a one-step decarboxylative condensation of malonyl-CoA and 4-coumaroyl-CoA to produce the diketide benzalacetone. We solved the crystal structures of both the wild-type and chalcone-producing I207L/L208F mutant of Rheum palmatum BAS at 1.8 Å resolution. In addition, we solved the crystal structure of the wild-type enzyme, in which a monoketide coumarate intermediate is covalently bound to the catalytic cysteine residue, at 1.6 Å resolution. This is the first direct evidence that type III PKS utilizes the cysteine as the nucleophile and as the attachment site for the polyketide intermediate. The crystal structures revealed that BAS utilizes an alternative, novel activesite pocket for locking the aromatic moiety of the coumarate, instead of the chalcone synthase's coumaroyl-binding pocket, which is lost in the active-site of the wild-type enzyme and restored in the I207L/L208F mutant. Furthermore, the crystal structures indicated the presence of a putative nucleophilic water molecule which forms hydrogen bond networks with the Cys-His-Asn catalytic triad. This suggested that BAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.biosynthesis | enzyme | polyketide