Biomedical subjects

M Wilmanns

Publications and source records attributed to M Wilmanns.

5 recordsLinked to original sources

PubMed1992

Protein engineering of a disulfide bond in a beta/alpha-barrel protein.

A disulfide bond has been introduced in the beta/alpha-barrel enzyme N-(5'-phosphoribosyl)anthranilate isomerase from Saccharomyces cerevisiae. The design of this disulfide bond was based on a model structure of this enzyme, built from the high-resolution crystal structure of the N-(5'-phosphoribosyl)anthranilate isomerase domain from Escherichia coli. The disulfide cross-link is spontaneously formed in vitro between residues 27 and 212, located in the structurally adjacent alpha-helices 1 and 8 of the outer helical ring of the beta/alpha-barrel. It creates a loop of 184 residues that account for 83% of the sequence of this enzyme, thus forming a quasi circular protein. The cross-linked mutant enzyme displays wild-type steady-state kinetic parameters. Measurements of the equilibrium constant for the reduction of this disulfide bond by 1,4-dithiothreitol show that its bond strength is comparable to that of other engineered protein disulfide bonds. The oxidized, cross-linked N-(5'-phosphoribosyl)anthranilate isomerase mutant is about 1.0 kcal/mol more stable than the wild-type enzyme, as estimated from its equilibrium unfolding transitions by guanidine hydrochloride.

Aldose-Ketose Isomerases↗

PubMed1992

Three-dimensional structure of the bifunctional enzyme phosphoribosylanthranilate isomerase: indoleglycerolphosphate synthase from Escherichia coli refined at 2.0 A resolution.

The three-dimensional structure of the monomeric bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli has been refined at 2.0 A resolution, using oscillation film data obtained from synchrotron radiation. The model includes the complete protein (452 residues), two phosphate ions and 628 water molecules. The final R-factor is 17.3% for all observed data between 15 and 2 A resolution. The root-mean-square deviations from ideal bond lengths and bond angles are 0.010 A and 3.2 degrees, respectively. The structure of N-(5'-phosphoribosyl)anthranilate isomerase: indole-3-glycerol-phosphate synthase from E. coli comprises two beta/alpha-barrel domains that superimpose with a root-mean-square deviation of 2.03 A for 138 C alpha-pairs. The C-terminal domain (residues 256 to 452) catalyses the PRAI reaction and the N-terminal domain (residues 1 to 255) catalyses the IGPS reaction, two sequential steps in tryptophan biosynthesis. The enzyme has the overall shape of a dumb-bell, resulting in a surface area that is considerably larger than normally observed for monomeric proteins of this size. The active sites of the PRAI and the IGPS domains, both located at the C-terminal side of the central beta-barrel, contain equivalent binding sites for the phosphate moieties of the substrates N-(5'-phosphoribosyl) anthranilate and 1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate. These two phosphate binding sites are identical with respect to their positions within the tertiary structure of the beta/alpha-barrel, the conformation of the residues involved in phosphate binding and the hydrogen-bonding network between the phosphate ions and the protein. The active site cavities of both domains contain similar hydrophobic pockets that presumably bind the anthranilic acid moieties of the substrates. These similarities of the tertiary structures and the active sites of the two domains provide evidence that N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from E. coli results from a gene duplication event of a monomeric beta/alpha-barrel ancestor.

Aldose-Ketose Isomerases↗

PubMed1991

Structural conservation in parallel beta/alpha-barrel enzymes that catalyze three sequential reactions in the pathway of tryptophan biosynthesis.

Three successive steps in tryptophan biosynthesis are catalyzed by single-domain proteins, each folded as a parallel beta/alpha-barrel, as observed in the crystal structures of the bienzyme (phosphoribosyl)-anthranilate isomerase:indoleglycerolphosphate synthase from Escherichia coli [Priestle, J.P., Grütter, M. G., White, J. L., Vincent, M. G., Kania, M., Wilson, E., Jardetzky, T. S., Kirschner, K., & Jansonius, J. N. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5690-5694] and the alpha-subunit of the tetrameric bienzyme tryptophan synthase from Salmonella typhimurium [Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., & Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871]. Recent refinement of the crystal structures of these enzymes at atomic resolution revealed that they contain a common phosphate group binding site in the beta/alpha-barrel, created by residues of the loop between beta-strand 7 and alpha-helix 7 and the N-terminus of an additional helix 8'. The close similarities of their beta/alpha-barrel structures permitted the alignment of 50-75% of their respective amino acid sequences. Considerable sequence similarity was detected in the regions spanning the phosphate binding sites, whereas the percentage of identical residues was barely significant for the remaining parts of the enzymes. These observations suggest divergent evolution of these three beta/alpha-barrel enzymes involved in tryptophan biosynthesis. The same phosphate binding site was also observed in six other beta/alpha-barrel enzymes that are functionally unrelated to those involved in tryptophan biosynthesis: triosephosphate isomerase, ribulose-1,5-bisphosphate carboxylase/oxygenase, glycolate oxidase, flavocytochrome b2, trimethylamine dehydrogenase, and tentatively also fructosebisphosphate aldolase.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldose-Ketose Isomerases↗

PubMed1990

Crystallization and structure solution at 4 A resolution of the recombinant synthase domain of N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli complexed to a substrate analogue.

The recombinant synthase domain of the bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli has been crystallized, and the structure has been solved at 4 A resolution. Two closely related crystal forms grown from ammonium sulphate diffract to 2 A resolution. One form (space group R32, a = 163 A, alpha = 29.5 degrees) contains the unliganded synthase domain; the second crystal form (space group P6(3)22, a = 144 A, c = 158 A) is co-crystallized with the substrate analogue N-(5'-phosphoribit-1-yl)anthranilate. The structure of the synthase-inhibitor complex has been solved by the molecular replacement method. This achievement represents the first successful use of a (beta alpha)8-barrel monomer as a trial model. The recombinant synthase domain associates as a trimer in the crystal, the molecules being related by a pseudo-crystallographic triad. The interface contacts between the three domains are mediated by those residues that are also involved in the domain interface of the bifunctional enzyme. This system provides a model for an interface which is used in both intermolecular and intramolecular domain contacts.

Aldose-Ketose Isomerases↗

PubMed1988

Amino acid sequence and three-dimensional structure of cytosolic adenylate kinase from carp muscle.

The amino acid sequence of cytosolic adenylate kinase from carp muscle has been determined. For the analysis of the blocked amino terminus an N-acylaminoacyl peptidase has been isolated and applied successfully. The resulting sequence is homologous to those of other adenylate kinases. It has 76% and 74% identical amino acids when compared with the enzymes from chicken and pig, respectively. The carp enzyme forms X-ray-grade crystals, the structure of which has been solved at 0.58-nm resolution. The observed chain fold is very similar to the known fold of the porcine enzyme in crystal form-A, whereas the molecular packing arrangements are quite different. This finding confirms that the observed porcine enzyme structure is intrinsically stable and has not been enforced during the crystallization process. The sequence of cytosolic adenylate kinase from carp muscle is: (table; see text)

Adenylate Kinase↗