Emil H. Harutyunyan

X‐Ray crystallographic studies of recombinant inorganic pyrophosphatase from Escherichia coli

An E. coli inorganic pyrophosphatase overproducer and a method for a large‐scale production of the homogeneous enzyme are described. The inorganic pyrophosphatase was crystallized in the form containing one subunit of a homohexameric molecule per asymmetric unit: space group R32, a = 110.4 Å, c = 76.8 Å. The electron density map to 2.5 Å resolution phased with Eu‐ and Hg‐derivatives (figure of merit, = 0.51) was improved by the solvent flattening procedure ( = 0.77). The course of the polypeptide chain and the secondary structure elements, intersubunit contacts and positions of the active sites were characterized. Homology with S. cerevisiae inorganic pyrophosphatase structure was found.

Escherichia coli inorganic pyrophosphatase : site-directed mutagenesis of the metal binding sites

Aspartic acids 65, 67, 70, 97 and 102 in the inorganic pyrophosphatase of Escherichia coli, identified as evolutionarily conserved residues of the active site, have been replaced by asparagine. Each mutation was found to decrease the κ app value by approx. 2–3 orders of magnitude. At the same time, the K m values changed only slightly. Only minor changes take place in the pK values of the residues essential for both substrate binding and catalysis. All mutant variants have practically the same affinity to Mg2+ as the wild‐type pyrophosphatase.

Crystal structure of Escherichia coli inorganic pyrophosphatase complexed with SO4(2-). Ligand-induced molecular asymmetry.

The three‐dimensional structure of inorganic pyrophosphatase from Escherichia coli complexed with sulfate was determined at 2.2 Å resolution using Pattersons search technique and refined to an R‐factor of 19.2%. Sulfate may be regarded as a structural analog of phosphate, the product of the enzyme reaction, and as a structural analog of methyl phosphate, the irreversible inhibitor. Sulfate binds to the pyrophosphatase active site cavity as does phosphate and this diminishes molecular symmetry, converting the homohexamer structure form (α3)2 into α3′α3″. The asymmetry of the molecule is manifested in displacements of protein functional groups and some parts of the polypeptide chain and reflects the interaction of subunits and their cooperation. The significance of re‐arrangements for pyrophosphatase function is discussed.

Mg2+ activation of Escherichia coli inorganic pyrophosphatase

Further refinement of X‐ray data on Escherichia coli inorganic pyrophosphatase [Oganessyan et al. (1994) FEBS Lett. 348, 301–304] to 2.2 Å reveals a system of noncovalent interactions involving Tyr55 and Tyr141 in the active site. The pKa for one of the eight Tyr residues in wild‐type pyrophosphatase is as low as 9.1 and further decreases to 8.1 upon Mg2+ binding, generating characteristic changes in the absorption spectrum. These effects are lost in a Y55F but not in a Y141F variant. It is suggested that the lower‐affinity site for Mg2+ in the enzyme is formed by Tyr55 and Asp70, which are in close proximity in the apo‐enzyme structure.

The polypeptide chain fold in tyrosine phenol-lyase, a pyridoxal-5′-phosphate-dependent enzyme

The tyrosine phenol lyase (EC 4.1.99.2) from Citrobacter intermedius has been crystallised in the apo form by vapour diffusion. The space group is P21212. The unit cell has dimensions a = 76.0 Å, b = 138.3 Å, c = 93.5 Å and it contains two subunits of the tetrameric molecule in the asymmetric unit, Diffraction data for the native enzyme and two heavy atom derivatives have been collected with synchrotron radiation and an image plate scanners. The structure has been solved at 2.7 Å resolution by isomorphous replacement with subsequent modification of the phases by averaging the density around the non‐crystallographic symmetry axis. The electron density maps clearly show the relative orientation of the subunits and most of the trace of the polypeptide chain. Each subunit consists of two domains. The topology of the large domain appears to be similar to that of the aminotransferases.

Crystal structure of thermitase from Thermoactinomyces vulgaris at 2.2 Å resolution

The crystal structure of thermitase from Thermoactinomyces vulgaris has been determined by X‐ray diffraction at 2.2 Å resolution. The structure was solved by a combination of single isomorphous replacement and molecular replacement methods. The structure was refined to a conventional R factor of 0.24 using restrained least square procedures CORELS and PROLSQ. The tertiary structure of thermitase is similar to that of subtilisin BPN′. The greatest differences between these structures are related to the insertions and deletions in the sequence.

Crystal structure of Escherichia coli inorganic pyrophosphatase complexed with SO4 2

The three‐dimensional structure of inorganic pyrophosphatase from Escherichia coli complexed with sulfate was determined at 2.2 Å resolution using Pattersons search technique and refined to an R‐factor of 19.2%. Sulfate may be regarded as a structural analog of phosphate, the product of the enzyme reaction, and as a structural analog of methyl phosphate, the irreversible inhibitor. Sulfate binds to the pyrophosphatase active site cavity as does phosphate and this diminishes molecular symmetry, converting the homohexamer structure form (α3)2 into α3′α3″. The asymmetry of the molecule is manifested in displacements of protein functional groups and some parts of the polypeptide chain and reflects the interaction of subunits and their cooperation. The significance of re‐arrangements for pyrophosphatase function is discussed.

Crystallization and preliminary X-ray studies of D-serine dehydratase from Escherichia coli.

Single crystals of D-serine dehydratase from Escherichia coli complexed with 3-amino-2-hydroxypropionate have been obtained from ammonium sulfate solution (pH 7.0) by vapor diffusion. The crystals belong to the trigonal space group P3(1) or P3(2) with a = b = 81.3 A and c = 58.4 A. The asymmetric unit cell contains one protein molecule with Mr = 48,289. The crystals diffract to at least 3.0 A resolution and are suitable for X-ray structure analysis.

A crystallographic station for structural investigations of macromolecular crystals on the synchrotron beam of the VEPP-3 storage ring

Abstract A station designed to speed up the collection of integral intensity data from protein and virus crystals is constructed on the synchrotron radiation beam of the VEPP-3 storage ring. A flat triangular Si crystal cut at 8° relative to the (111) plane is used to monochromatize the radiation. A conventional Arndt-Wonacott rotation camera is used to collect structural information. The station also includes an optical bench which can turn about the axis coinciding with that of the monochromator, and a set of slits to cut the background and to form the beam. The ionization chamber serves to adjust the monochromator and to monitor the beam. Numerical results of first measurements from protein crystals as well as plans for future work are presented.

Crystallization and crystal data on tyrosine phenol‐lyase

Crystals of the apoenzyme of tyrosine phenol‐lyase (EC 4.1.99.2), a pyridoxal 5′‐phosphate‐dependent enzyme from Citrobacter intermedius, have been grown by vapor diffusion of an ammonium sulfate solution to a protein solution. The crystals belong to space group P21212, with dimensions of a = 75.5 Å, b = 138.4 Å and c = 94.1 Å and diffract up to 2.7 Å resolution. The asymmetric unit contains one half of the enzyme tetrameric molecule. Two heavy‐atom derivatives of the crystals have been obtained.