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D Alexeev

Publications and source records attributed to D Alexeev.

12 recordsLinked to original sources
PubMed2000

Mechanism of 8-amino-7-oxononanoate synthase: spectroscopic, kinetic, and crystallographic studies.

8-Amino-7-oxononanoate synthase (also known as 7-keto-8-aminopelargonate synthase, EC 2.3.1.47) is a pyridoxal 5'-phosphate-dependent enzyme which catalyzes the decarboxylative condensation of L-alanine with pimeloyl-CoA in a stereospecific manner to form 8(S)-amino-7-oxononanoate. This is the first committed step in biotin biosynthesis. The mechanism of Escherichia coli AONS has been investigated by spectroscopic, kinetic, and crystallographic techniques. The X-ray structure of the holoenzyme has been refined at a resolution of 1.7 A (R = 18.6%, R(free) = 21. 2%) and shows that the plane of the imine bond of the internal aldimine deviates from the pyridine plane. The structure of the enzyme-product external aldimine complex has been refined at a resolution of 2.0 A (R = 21.2%, R(free) = 27.8%) and shows a rotation of the pyridine ring with respect to that in the internal aldimine, together with a significant conformational change of the C-terminal domain and subtle rearrangement of the active site hydrogen bonding. The first step in the reaction, L-alanine external aldimine formation, is rapid (k(1) = 2 x 10(4) M(-)(1) s(-)(1)). Formation of an external aldimine with D-alanine, which is not a substrate, is significantly slower (k(1) = 125 M(-)(1) s(-)(1)). Binding of D-alanine to AONS is enhanced approximately 2-fold in the presence of pimeloyl-CoA. Significant substrate quinonoid formation only occurs upon addition of pimeloyl-CoA to the preformed L-alanine external aldimine complex and is preceded by a distinct lag phase ( approximately 30 ms) which suggests that binding of the pimeloyl-CoA causes a conformational transition of the enzyme external aldimine complex. This transition, which is inferred by modeling to require a rotation around the Calpha-N bond of the external aldimine complex, promotes abstraction of the Calpha proton by Lys236. These results have been combined to form a detailed mechanistic pathway for AONS catalysis which may be applied to the other members of the alpha-oxoamine synthase subfamily.

Acyl Coenzyme A↗
PubMed1999

Crystallization and preliminary X-ray analysis of flavocytochrome c(3), the fumarate reductase from Shewanella frigidimarina.

The fumarate reductase (flavocytochrome c(3)) from Shewanella frigidimarina (formerly S. putrefaciens) NCIMB400 has been crystallized in the space group P2(1), with cell dimensions of a = 45.447 A, b = 92.107 A, c = 78.311 A, and beta = 91.038 degrees and one molecule per asymmetric unit. A native data set has been collected to 1.8 A. The gene encoding Fcc(3) from the S. frigidimarina type strain ACAM591 has been cloned and sequenced and the protein crystallized in space group P2(1) with cell dimensions of a = 45.359 A, b = 88.051 A, c = 77.473 A, and beta = 104.499 degrees. Anomalous data have also been collected from the NCIMB400 crystal allowing the heme iron positions to be identified.

Base Sequence↗
PubMed1998

The crystal structure of 8-amino-7-oxononanoate synthase: a bacterial PLP-dependent, acyl-CoA-condensing enzyme.

8-Amino-7-oxononanoate synthase (or 8-amino-7-ketopelargonate synthase; EC 2.3.1.47; AONS) catalyses the decarboxylative condensation of l-alanine and pimeloyl-CoA in the first committed step of biotin biosynthesis. We have cloned, over-expressed and purified AONS from Escherichia coli and determined the crystal structures of the apo and PLP-bound forms of the enzyme. The protein is a symmetrical homodimer with a tertiary structure and active site organisation similar to, but distinct from, those of other PLP-dependent enzymes whose three-dimensional structures are known. The critical PLP-binding lysine of AONS is located at the end of a deep cleft that allows access of the pantothenate arm of pimeloyl-CoA. A cluster of positively charged residues at the entrance to this cleft forms a putative diphosphate binding site for CoA. The structure of E. coli AONS enables identification of the key residues of the PLP-binding site and thus provides a framework with which to understand the biochemical mechanism, which is similar to that catalysed by 5-aminolevulinate synthase and two other alpha-oxoamine synthases. Although AONS has a low overall sequence similarity with the catalytic domains of other alpha-oxoamine synthases, the structure reveals the regions of significant identity to be functionally important. This suggests that the organisation of the conserved catalytic residues in the active site is similar for all enzymes of this sub-class of PLP-dependent enzymes and they share a common mechanism. Knowledge of the three-dimensional structure of AONS will enable characterisation of the structural features of this enzyme sub-family that are responsible for this important type of reaction.

Acyl Coenzyme A↗
PubMed1998

The 2.3 A X-ray crystal structure of S. cerevisiae phosphoglycerate mutase.

The high resolution crystal structure of Saccharomyces cerevisiae phosphoglycerate mutase has been determined. This structure shows important differences from the lower resolution structure deposited in 1982. The crystal used to determine the new structure was of a different form, having spacegroup P2(1). The model was refined to a crystallographic R-factor of 18.9% and a free R-factor of 28.4% using all data between 25 and 2.3 A and employing a bulk solvent correction. The enzyme is a tetramer of identical, 246 amino acid subunits, whose structure is revealed to be a dimer of dimers, with four independent active sites located well away from the subunit contacts. Each subunit contains two domains, the larger with a typical nucleotide binding fold, although phosphoglycerate mutase has no physiological requirement to bind nucleotides. The catalytic-site histidine residues are no longer in a "clapping-hands" conformation, but more resemble the conformation seen in the distantly related enzymes prostatic acid phosphatase and fructose-2,6-bisphosphatase. However, the catalytic histidine residues in the mutase are found to be much closer to each other than in the phosphatase structures, perhaps due to the absence of bound ligands in the mutase crystal. An intricate web of H-bonds is found around the catalytic histidine residues, high-lighting residues probably important for maintaining their correct orientation and charge. The positions of certain other residues, including some found near the catalytic site and some lining the catalytic-site cleft, have been changed by the correction of registration errors between sequence and electron density in the original structure. Electron density was apparent for a portion of the functionally important C-terminal tail, which was absent from the earlier structure, showing it to adopt a mainly helical conformation.

Acid Phosphatase↗
PubMed1997

Synthetic, structural and biological studies of the ubiquitin system: synthesis and crystal structure of an analogue containing unnatural amino acids.

Ubiquitin is a 76-amino acid protein involved in the targeting for destruction of proteins in the cell. The protein can readily be synthesized chemically affording an extra dimension to studies of protein stability. Ubiquitin with various modifications to the hydrophobic core has been synthesized. In particular, two core amino acids have been replaced by aminobutyric acid (Val-26) and norvaline (for Ile-30) and the product crystallized. The refined crystal structure shows an overall contraction of the molecule and the side chain of Nva-30 rotates relative to Ile-30. However, the side chain rotation is not sufficient to compensate for the effect of the loss of the methyl group and hence a small cavity is introduced into the structure, which decreases the stability of the protein. The biological behaviour of the modified protein is unaltered. The observed changes in stability are of the magnitude expected for the removal of methyl groups from the hydrophobic core of a protein. Interestingly, the effect appears to be independent of the position of the removed methyl group. The intact structure, but not its stability, is important for recognition by the biological conjugating system.

Aminobutyrates↗
PubMed1995

Substrate binding and carboxylation by dethiobiotin synthetase--a kinetic and X-ray study.

BACKGROUND: The vitamin biotin is a ubiquitous prosthetic group of carboxylase and transcarboxylase enzymes. Biotin biosynthesis occurs by similar pathways in microorganisms and plants. The penultimate step in biotin biosynthesis, catalyzed by dethiobiotin synthetase (DTBS), involves a unique ATP-dependent N-carboxylation, resulting in formation of the ureido ring function of dethiobiotin. The first two steps of dethiobiotin formation, which is a complex, multistep enzymatic reaction, have been elucidated by a combination of X-ray crystallography and kinetic methods. RESULTS: The first step in catalysis by DTBS is the formation of an enzyme-substrate complex and the second is the enzymatic carboxylation of the bound substrate. Both steps are Mg2+ dependent. The kinetic constants in the presence and absence of Mg2+ have been measured and a set of X-ray structures determined at different stages of the reaction. The conformational changes in the active site of the enzyme, induced by Mg2+, substrate binding and substrate carboxylation, have been monitored crystallographically and are discussed. Sulfate ions bound to DTBS may mimic the behaviour of the alpha- and gamma-phosphates of ATP in Mg2+ binding and in the subsequent steps of the reaction. CONCLUSIONS: Mg2+ is an essential cation for both substrate binding and carbamate formation by DTBS, when sulfate is present. The conformational changes induced at the active site in the DTBS-substrate complex, when Mg2+ is present, are small yet highly significant and serve to optimize the interactions between substrate and enzyme. DTBS is active as a homodimer and the substrate-binding site straddles both monomers in the dimer. The carboxylation site is unambiguously identified as the N-7 amino group of the substrate, rather than the N-8 amino group, as previously suggested. The elongated nucleotide-binding loop (the P loop) binds both ATP and substrate in a manner which suggests that this feature may be of wider importance.

Acylation↗
PubMed1995

Correlation function method in protein crystallography.

A weighted correlation function as a method for computing electron-density maps is proposed to reduce the errors of the Fourier syntheses performed on inaccurate and/or incomplete data. The formulae are revised for the difference Patterson vector search, for multiple isomorphous replacement (MIR) and single isomorphous replacement (SIR) syntheses and for the difference Fourier synthesis. The examples show that the correlation-function approach has the potential to provide more reliable results than those obtained by conventional Fourier syntheses.

Journal Article↗
PubMed1994

Mechanistic implications and family relationships from the structure of dethiobiotin synthetase.

BACKGROUND: Biotin is the vitamin essential for many biological carboxylation reactions, such as the conversion of acetyl-coenzyme A (CoA) to malonyl-CoA in fatty acid biosynthesis. Dethiobiotin synthetase (DTBS) facilitates the penultimate, ureido ring closure in biotin synthesis, which is a non-biotin-dependent carboxylation. DTBS displays no sequence similarity to any other protein in the database. Structural studies provide a molecular insight into the reaction mechanism of DTBS. RESULTS: We present the structure of DTBS refined to 1.80 A resolution with an R-factor of 17.2% for all terms plus unrefined data on the binding of the substrate, 7,8-diaminopelargonic acid and the product, dethiobiotin. These studies confirm that the protein forms a homodimer with each subunit folded as a single globular alpha/beta domain. The presence of sulphate ions in the crystals and comparisons with the related Ha-ras-p21 oncogene product are used to infer the ATP-binding site, corroborated by the difference electron density for the ATP analogue AMP-PNP. CONCLUSIONS: This study establishes that the enzyme active site is situated at the dimer interface, with the substrate binding to one monomer and ATP to the other. The overall fold of DTBS closely resembles that of three other enzymes, adenylosuccinate synthetase (purA), Ha-ras-p21, and nitrogenase iron protein, that are unrelated by sequence or function, indicating that DTBS is a member of a diverse family of enzymes.

Adenosine Triphosphate↗
PubMed1994

Synthetic, structural and biological studies of the ubiquitin system: chemically synthesized and native ubiquitin fold into identical three-dimensional structures.

The solid-phase chemical synthesis of ubiquitin produced a molecule with physicochemical properties similar to those of the natural protein. We have grown crystals of this synthetic ubiquitin and performed an X-ray analysis at 1.8 A resolution in order to compare the synthetic protein with the known natural structure. The crystals were isomorphous with those of the natural protein, the R-factor between them being 7.1%. Difference Fourier analysis shows that the synthetic and natural structures are indistinguishable. The co-ordinates of the natural ubiquitin (1UBQ) were used as the starting point for restrained least-squares refinement (TNT program) against the synthetic X-ray data. The refinement converged to R = 16.5% and the resulting model did not change when refined against natural ubiquitin X-ray data (R = 18.7%). From both the refinement and featureless difference Fourier synthesis, we conclude that the synthetic and natural protein structures are identical. A short discussion about the uses of proteins with 'non-standard' amino acid residues is included.

Crystallography, X-Ray↗
PubMed1994

Sequence and crystallization of Escherichia coli dethiobiotin synthetase, the penultimate enzyme of biotin biosynthesis.

The enzyme dethiobiotin synthetase (EC 6.3.3.3) has been cloned and over-expressed in Escherichia coli in such a way that milligram quantities are available. The purified enzyme has been subjected to a number of physical and chemical studies, sequenced and most notably it has been crystallized in a form that is suitable for X-ray structure determination. The cell dimensions are a = 72.8 A, b = 49.2 A, c = 61.4 A, beta = 106.2 degrees. The systematic absences are consistent with the monoclinic space group C2 with one polypeptide chain in the asymmetric unit.

Amino Acid Sequence↗