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F S Mathews

Publications and source records attributed to F S Mathews.

At least 91 records · Page 5Linked to original sources
PubMed1989

Studies of crystalline trimethylamine dehydrogenase in three oxidation states and in the presence of substrate and inhibitor.

Crystals of trimethylamine dehydrogenase have been examined by difference Fourier methods at 6.0-A resolution after partial reduction by substrate and by dithionite in the presence of inhibitor. Similar studies of the inhibited oxidized enzyme and of the enzyme reduced fully by dithionite alone were also carried out. In all cases ligand binding at the active site occurred. In addition, there were small structural changes, possibly side chain movements, in the inhibited oxidized enzyme and somewhat larger changes in the partially reduced crystals. The largest changes occurred with the fully reduced enzyme. However, in no cases were subunit or domain movements observed nor were changes observed in the position of the FMN or [4Fe-4S] cofactors. Parallel studies of crystalline trimethylamine dehydrogenase were carried out by EPR spectroscopy. The results show that the electronic states of the crystalline enzyme under the conditions of the difference Fourier studies are comparable to those which occur in solution under similar conditions.

Crystallization↗
PubMed1988

Crystallographic study of the complex between sulfite and bakers' yeast flavocytochrome b2.

The complex between Saccharomyces cerevisiae flavocytochrome b2 and the sulfite anion has been analyzed by x-ray diffraction. A map of the difference in electron density between the complex and the native protein has been computed. One positive peak of electron density is visible at the active site of each of the two subunits in the asymmetric unit, very close to the N-5 of the flavin. The molecular fragment SO3(2-) can account for the shape of this difference in electron density. A third peak is visible in the subunit containing pyruvate, the reaction product. It is a peak of negative electron density localized at the position where the pyruvate usually is in the native form. These results are interpreted on the basis of the mechanism defined in solution for the reaction between flavins and sulfite.

Binding Sites↗
PubMed1988

Probing the active site of flavocytochrome b2 by site-directed mutagenesis.

The three-dimensional structure of flavocytochrome b2 (L-lactate dehydrogenase) from bakers' yeast (Saccharomyces cerevisiae) has recently been solved at 0.24-nm resolution [Mathews & Xia (1987) in Flavins and flavoproteins, Walter de Gruyter, Berlin, pp. 123-131]. We have used this structural information to investigate the roles of particular amino acid residues likely to be involved in the oxidation of L-lactate by kinetic analysis of mutant enzymes generated by site-directed mutagenesis of the isolated gene. The hydroxyl group of Tyr254 was expected to be important for the abstraction of the hydroxyl proton of L-lactate in the oxidation to pyruvate. Replacement of this tyrosine by phenylalanine reduced kcat from 190 +/- 3 s-1 (25 degrees C, pH 7.5) to 4.3 +/- 0.1 s-1. This substitution had, however, no discernable effect on Km for lactate (0.54 +/- 0.03 mM for the mutant compared with 0.49 +/- 0.03 mM for the wild-type enzyme). Arg376 was expected to be essential for productive binding and orientation of L-lactate. Replacing Arg376 with lysine abolished all detectable activity. A total loss of enzymic activity was also observed when Lys349, thought likely to stabilize the anionic form of the flavin hydroquinone, was replaced by arginine. An amino acid residue replacement at a distance from the active site, Ala306 to serine, had a minor but significant effect on kcat (reduced from 190 s-1 to 160 s-1) and Km (increased from 0.49 mM to 0.83 mM) presumably arising from small conformational effects. The implications of these results are discussed in relation to the mechanism of L-lactate oxidation.

Binding Sites↗
PubMed1988

Identification of ADP in the iron-sulfur flavoprotein trimethylamine dehydrogenase.

Analysis of the 2.4-A resolution electron density map of trimethylamine dehydrogenase has revealed the unexpected presence of one molecule of ADP/subunit. This binding has been confirmed chemically. The binding site is located at the analogous position of the ADP moiety of FAD in glutathione reductase, the FAD and NADPH binding domains of which resemble two of the domains of trimethylamine dehydrogenase. Comparison of the environments of the ADP moieties in the two proteins indicates that 32 residues in 6 peptides are in equivalent positions with a root mean square deviation for C alpha positions of 1.11 A. Twelve of these amino acids are identical, based on the electron density-derived "x-ray" sequence of trimethylamine dehydrogenase. Detailed analysis of the environment of the ADP moiety indicates that most of the conserved residues are not in direct contact with the cofactor. Some of them probably represent the "fingerprint" of the beta alpha beta binding fold found in dinucleotide binding proteins, but the remaining conserved residues may indicate a closer evolutionary relationship between these two proteins.

Adenosine Diphosphate↗
PubMed1987

Three-dimensional structure of flavocytochrome b2 from baker's yeast at 3.0-A resolution.

The structure of flavocytochrome b2 from baker's yeast was solved at 3.0-A resolution by the multiple isomorphous replacement method combined with solvent leveling procedures, using data collected from an area detector. The tetramer of Mr 230,000 has 4-fold symmetry. Each subunit contains a cytochrome domain consisting of the first 100 residues, a flavin-binding domain containing the next 386 residues, and an extended C-terminal tail of 25 residues. The cytochrome domain closely resembles microsomal cytochrome b5, whereas the flavin-binding domain contains a parallel beta 8/alpha 8 barrel motif similar to glycolate oxidase and trimethylamine dehydrogenase. Two of the four cytochrome domains are disordered in the crystals. The flavin ring and heme group are separated by about 16 A between their centers, and their planes are inclined by about 17 degrees to each other.

L-Lactate Dehydrogenase↗
PubMed1986

Three-dimensional structure of the iron-sulfur flavoprotein trimethylamine dehydrogenase at 2.4-A resolution.

The three-dimensional structure of trimethylamine dehydrogenase from the methylotrophic bacterium W3A1 has been determined to 2.4-A resolution. The enzyme is composed of two identical 83,000-dalton subunits, each of which is folded into three structural domains. The largest domain, at the NH2 terminus of the molecule, is folded as an eight-stranded parallel alpha/beta barrel. It contains the [4Fe-4S] and covalently bound FMN cofactors separated by about 4 A. The folding topology of the large domain and orientation of the FMN cofactor are very similar to those found in glycolate oxidase. The other two domains contain alpha/beta parallel beta sheet topologies with similar folding patterns. The topologies and spatial arrangements of these two domains are remarkably similar to the FAD- and NADPH-binding domains of glutathione reductase.

Bacteria↗
PubMed1986

Amino acid and sequence analysis of the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase.

The flavocytochrome p-cresol methylhydroxylase from Pseudomonas putida has been reported to have a Mr of 114,000 and to consist of two subunits, a flavoprotein and a cytochrome c, each with a Mr of 58,000. Recent X-ray crystallographic data from our laboratories [Shamala, N., Lim, L. W., Mathews, F. S., McIntire, W., Singer, T. P., & Hopper, D. J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4626-4630], however, indicate an alpha 2 beta 2 structure and a much lower molecular mass (approximately 8000) for the cytochrome subunit. In this paper we report data confirming the conclusions of X-ray crystallographic analysis. From quantitative amino acid analysis, the molecular mass of the flavoprotein monomer is shown to be 48,600 +/- 2200 and that of the cytochrome 8780 +/- 250. These values have been confirmed by gel electrophoresis under denaturing conditions. Gel chromatography under nondenaturing conditions shows that the isolated flavoprotein exists as a dimer, whereas the isolated cytochrome is a monomer. The complete amino acid sequence of the cytochrome c subunit is presented and is shown to have regions of homology to other bacterial c-type cytochromes. The partial N-terminal amino acid sequence (56 amino acids) of the flavoprotein subunit is also reported. The implications of the now established tetrameric structure of the flavocytochrome on data in the literature regarding the redox and association properties of the subunits are examined.

Amino Acid Sequence↗
PubMed1986

Preliminary X-ray crystallographic study of methanol dehydrogenase from Methylophilus methylotrophus.

Single crystals of methanol dehydrogenase from Methylophilus methylotrophus have been prepared by the macroseeding method. The crystals belong to the monoclinic space group C2, and have unit cell parameters a = 125.62 A, b = 63.83 A, c = 83.99 A, and beta = 93.24 degrees. There is one 62,000 Mr monomer in the asymmetric unit. The crystals diffract to beyond 2.0 A resolution.

Alcohol Oxidoreductases↗
PubMed1986

Preliminary X-ray crystallographic study of amicyanin from Paracoccus denitrificans.

Single crystals have been prepared of Paracoccus denitrificans amicyanin, a blue copper protein that serves as an electron acceptor for methylamine dehydrogenase. The crystals belong to the monoclinic space group P2(1), and have unit cell parameters a = 20.90 A, b = 56.61 A, c = 27.55 A and beta = 96.41. There is one molecule in the asymmetric unit. The crystals diffract to beyond 1.5 A resolution.

Bacterial Proteins↗
PubMed1986

Structure of an intermolecular electron-transfer complex: p-cresol methylhydroxylase at 6.0-A resolution.

The structure of p-cresol methylhydroxylase [4-cresol:(acceptor) oxidoreductase (methyl-hydroxylating), EC 1.17.99.1], a flavocytochrome c, has been determined at 6.0-A resolution. The structure analysis is based on two heavy-atom derivatives with anomalous scattering and 2-fold averaging about a noncrystallographic axis. The molecule is an alpha 2 beta 2 tetramer with a cytochrome subunit of Mr approximately 8500 and a flavoprotein subunit of Mr approximately 49,000. The flavoprotein subunits are tightly packed about the molecular 2-fold axis, whereas the cytochrome subunits are located on the outside of the molecule, each in a depression on the surface of a flavoprotein subunit. The results of this study have led to the following conclusions. The alpha 2 beta 2 quaternary structure of the enzyme is different from alpha beta as originally thought. The orientation of the cytochrome subunit and the surface complementarity of the cytochrome and flavoprotein subunits are clearly defined. The cytochrome subunit is similar in size to other small bacterial cytochromes but probably forms a distinct subclass. The titration (by substrate) behavior of the enzyme and other kinetic properties are rationalized by its quaternary structure.

Bacterial Proteins↗
PubMed1985

A spectroscopic investigation of the structure and redox properties of Escherichia coli cytochrome b-562.

The six-coordinate monohaem ferricytochrome b-562 from Escherichia coli exhibits two haem-linked pH-dependent transitions detected by NMR and optical spectroscopy. Only one of these transitions, that of the Fe(III)-coordinated His-102, is detected by EPR and MCD; the ionisation of a haem propionate is not. Both ionisations are redox-state-dependent and the midpoint redox potential of the protein is markedly pH-dependent. Over the pH range 5.0 to 8.5 the potential drops from 260 mV to 110 mV and at least five single proton ionisations are responsible for this. In addition to the two spectroscopically identified ferricytochrome ionisations, there are at least three unidentified ionisations, two of which occur in the ferrous protein. From a consideration of the X-ray structure, together with NMR data, it seems probable that at least one of these ionisations involves an amino acid carboxylate. The X-ray structure also suggests that the relatively low pKa of His-102 is a result of its proximity to Arg-98. However, an appreciable interaction between these groups requires that the solution conformation differs slightly from the X-ray structure. The fast rate of electron self-exchange, over 4 X 10(6) M-1 X s-1 at 315 K and pH* 7, may be a reflection of the fact that, as shown by the X-ray structure, a large amount of the haem and axial histidine ligand are exposed at the molecular surface with an asymmetric distribution of charged groups surrounding them.

Chemical Phenomena↗
PubMed1984

Molecular structure of trimethylamine dehydrogenase from the bacterium W3A1 at 6.0-A resolution.

An electron density map of trimethylamine dehydrogenase has been calculated at 6.0-A resolution. Protein phases were based on two isomorphous mercury derivatives with similar binding properties, and on anomalous scattering measurements. The map has been averaged about the noncrystallographic 2-fold axis, plotted on transparent sheets and used to construct a wooden model. The elipsoidal dimer has a large inter-subunit interface. Each subunit appears to contain three closely associated domains with the iron-sulfur cluster located between two of them. The map suggests an alpha/beta-structure for two of the domains and a large helix content for the third.

Bacteria↗
PubMed1983

Crystal and molecular structure of tert.-butyloxycarbonyl-L-prolyl-alpha-aminoisobutyryl-L-alanyl-alpha- aminoisobutyrate methyl ester.

Boc-Pro-Aib-Ala-Aib-OMe crystallizes in the orthorhombic space group P2(1)2(1)2 with cell dimensions a = 17.701 (3) A, b = 17.476 (4) A, c = 9.686 (2) A, V = 2996.3 A3. The first three residues form a single turn of a 3(10)-helix stabilized by two intramolecular hydrogen bonds. Comparison of the conformation of the methyl ester of the tetrapeptide with that of its benzyl ester shows differences in the individual torsion angles of up to 29 degrees, although the overall conformation is conserved.

Alamethicin↗
PubMed1981

On the evolutionary relationship of the 4-alpha-helical heme proteins. The comparison of cytochrome b562 and cytochrome c'.

The atomic models of the cytochrome b562 and cytochrome c' monomers have been compared. When the respective heme groups are superimposed, the four alpha-helices of each nearly coincide. Four aromatic side chains, including the heme ligands, and a methionine occur in spatially equivalent positions in contact with the heme groups. This structural evidence suggests that the two cytochrome families may have diverged from a common molecular ancestor.

Biological Evolution↗