Crystallization and structure solution at 4 Å 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 Å resolution. Two closely related crystal forms grown from ammonium sulphate diffract to 2 Å resolution. One form (space group R32, a = 163 Å, α = 29.5°) contains the unliganded synthase domain; the second crystal form (space group P6322, a = 144 Å, c = 158 Å) 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 (βα)g-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 contact

Three-dimensional structure of a mutant E.coli aspartate aminotransferase with increased enzymic activity

The aspartate and tyrosine aminotransferases from Escherichia coli have 43% sequence identity and nearly identical active sites. Both are equally good enzymes for dicarboxylate substrates, but the latter transaminates aromatic amino acids 1000 times faster. In an attempt to discover the critical residues for this differential substrate specificity, the aspartate aminotransferase mutant V39L has recently been prepared. It showed improved kcal/Km values for aspartate, glutamate and tyrosine and the corresponding oxo acids, mainly due to two to ten times lower Km values. For example, the Km values of V39L (wild type) for Asp and Glu are 0.12 (1.0) and 0.85 (2.7) mM respectively. The mutant was co-crystallized with 30 mM maleate from both polyethylene glycol and ammonium sulfate. Both structures were solved and refined to R-factors of 0.22 and 0.20 at 2.85 and 2.5 Å resolution respectively. They bear strong resemblance to the closed structure of the wild type enzyme complexed with maleate. The unexpected feature is that, for the first time, the closed form was produced in crystals grown from ammonium sulfate. It is concluded that the mutation has shifted the conformational equilibrium towards the closed form, which leads to generally reduced substrate Km

2.0 å structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability

AbstractBackground: Recent efforts to understand the basis of protein stability have focussed attention on comparative studies of proteins from hyperthermophilic and mesophilic organisms. Most work to date has been on either oligomeric enzymes or monomers comprising more than one domain. Such studies are hampered by the need to distinguish between stabilizing interactions acting between subunits or domains from those acting within domains. In order to simplify the search for determinants of protein stability we have chosen to study the monomeric enzyme indole-3-glycerol phosphate synthase from the hyperthermophilic archaeon Sulfolobus solfataricus (sIGPS), which grows optimally at 90°C.Results The 2.0 å crystal structure of sIGPS was determined and compared with the known 2.0 å structure of the IGPS domain of the bifunctional enzyme from the mesophilic bacterium Escherichia coli (eIGPS). sIGPS and eIGPS have only 30% sequence identity, but share high structural similarity. Both are single-domain (β/α)8 barrel proteins, with one (eIGPS) or two (sIGPS) additional helices inserted before the first β strand. The thermostable sIGPS has many more salt bridges than eIGPS. Several salt bridges crosslink adjacent α helices or participate in triple or quadruple salt-bridge clusters. The number of helix capping, dipole stabilizing and hydrophobic interactions is also increased in sIGPS.Conclusion The higher stability of sIGPS compared with eIGPS seems to be the result of several improved interactions. These include a larger number of salt bridges, stabilization of α helices and strengthening of both polypeptide chain termini and solvent-exposed loops

Crystal Structure of Concanavalin B at 1.65 Å Resolution. An ‘‘Inactivated’’ Chitinase from Seeds of Canavalia ensiformis

Seeds of Canavalia ensiformis (jack bean) contain besides large amounts of canavalin and concanavalin A, a protein with a molecular mass of 33,800 which has been named concanavalin B. Although concanavalin B shares about 40% sequence identity with plant chitinases belonging to glycosyl hydrolase family 18, no chitinase activity could be detected for this protein. To resolve this incongruity concanavalin B was crystallised and its three-dimensional structure determined at 1.65 Å (1 Å = 0.1 nm) resolution. The structure consists of a single domain with a (β/α)8 topology. A 30 amino acid residue long loop occurs between the second β-strand of the barrel and the second α-helix. This extended loop is unusual for the (β/α)8 topology, but appears in a similar conformation in the structures of the seed protein narbonin and several chitinases as well. Two non-proline cis-peptide bonds are present in the structure of concanavalin B: Ser34-Phe, and Trp265-Asn. This structural feature is rarely observed in proteins, but could also be identified in the three-dimensional structures of family 18 chitinases and narbonin in coincident positions. In the chitinases the aromatic residues of the non-proline cis-peptides have been proposed to have a function in the binding of the substrate. The region in concanavalin B, where in chitinases the active site is located, shows two significant differences. First, the catalytic glutamic acid is a glutamine in concanavalin B. Second, although part of the substrate binding cleft of the chitinases is present in concanavalin B, it is much shorter. From this we conclude that concanavalin B and family 18 chitinases are closely related, but that concanavalin B has lost its enzymatic function. It still may act as a carbohydrate binding protein, however.