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M L Ludwig

Publications and source records attributed to M L Ludwig.

At least 55 records · Page 3Linked to original sources
PubMed1991

Manganese superoxide dismutase from Thermus thermophilus. A structural model refined at 1.8 A resolution.

The structure of Mn(III) superoxide dismutase (Mn(III)SOD) from Thermus thermophilus, a tetramer of chains 203 residues in length, has been refined by restrained least-squares methods. The R-factor [formula: see text] for the 54,056 unique reflections measured between 10.0 and 1.8 A (96% of all possible reflections) is 0.176 for a model comprising the protein dimer and 180 bound solvents, the asymmetric unit of the P4(1)2(1)2 cell. The monomer chain forms two domains as determined by distance plots: the N-terminal domain is dominated by two long antiparallel helices (residues 21 to 45 and 69 to 89) and the C-terminal domain (residues 100 to 203) is an alpha + beta structure including a three-stranded sheet. Features that may be important for the folding and function of this MnSOD include: (1) a cis-proline in a turn preceding the first long helix; (2) a residue inserted at position 30 that distorts the helix near the first Mn ligand; and (3) the locations of glycine and proline residues in the domain connector (residues 92 to 99) and in the vicinity of the short cross connection (residues 150 to 159) that links two strands of the beta-sheet. Domain-domain contacts include salt bridges between arginine residues and acidic side chains, an extensive hydrophobic interface, and at least ten hydrogen-bonded interactions. The tetramer possesses 222 symmetry but is held together by only two types of interfaces. The dimer interface at the non-crystallographic dyad is extensive (1000 A2 buried surface/monomer) and incorporates 17 trapped or structural solvents. The dimer interface at the crystallographic dyad buries fewer residues (750 A2/monomer) and resembles a snap fastener in which a type I turn thrusts into a hydrophobic basket formed by a ring of helices in the opposing chain. Each of the metal sites is fully occupied, with the Mn(III) five-co-ordinate in trigonal bipyramidal geometry. One of the axial ligands is solvent; the four protein ligands are His28, His83, Asp166 and His170. Surrounding the metal-ligand cluster is a shell of predominantly hydrophobic residues from both chains of the asymmetric unit (Phe86A, Trp87A, Trp132A, Trp168A, Tyr183A, Tyr172B, Tyr173B), and both chains collaborate in the formation of a solvent-lined channel that terminates at Tyr36 and His32 near the metal ion and is presumed to be the path by which substrate or other inner-sphere ligands reach the metal.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

Structure-function relationships in iron and manganese superoxide dismutases.

Using the complete sequences for MnSOD from Thermus thermophilus and for FeSOD from E. coli, structural models for both oxidized enzymes have been refined, the Mn protein to an R of 0.186 for all data between 10.0 and 1.8 A, and the Fe protein to an R of 0.22 for data between 10.0 and 2.5 A. The results of the refinements support the presence of a solvent as a fifth ligand to Mn(III) and Fe(III) and a coordination geometry that is close to trigonal bipyramidal. The putative substrate-entry channel is comprised of residues from both subunits of the dimer; several basic residues that are conserved may facilitate approach of O2-, while other conserved residues maintain interchain packing interactions. Analysis of the azide complex of Fe(III) dismutase suggests that during turnover O2- binds to the metal at a sixth coordination site without displacing the solvent ligand. Because crystals reduced with dithionite show no evidence for displacement of the protein ligands, the redox-linked proton acceptor (C. Bull and J.A. Fee (1985), Journal of the American Chemistry Society 107, 3295-3304) is unlikely to be one of the histidines which bind the metal ion. Structural, kinetic, titration, and spectroscopic data can be accommodated in a mechanistic scheme which accounts for the differential titration behaviour of the Fe(III) and Fe(II) enzymes at neutral and high pH.

Amino Acid Sequence↗
PubMed1990

Structure and oxidation-reduction behavior of 1-deaza-FMN flavodoxins: modulation of redox potentials in flavodoxins.

Flavodoxins from Clostridium beijerinckii and from Megasphaera elsdenii with 1-carba-1-deaza-FMN substituted for FMN have been used to study flavin-protein interactions in flavodoxins. The oxidized 1-deaza analogue of FMN binds to apoflavodoxins from M. elsdenii and C. beijerinckii (a.k.a. Clostridium MP) with association constants (Ka) of 1.0 x 10(7) M-1 and 3.1 x 10(6) M-1, values about 10(2) less than the corresponding Ka values for FMN. X-ray structure analysis of oxidized 1-deaza-FMN flavodoxin from C. beijerinckii at 2.5-A resolution shows that the analogue binds with the flavin atoms in the same locations as their equivalents in FMN but that the protein moves in the vicinity of Gly 89 to accommodate the 1-CH group, undergoing displacements which increase the distance between position 1 of the flavin ring and the main-chain atoms of Gly 89 and move the peptide hydrogen of Gly 89 by about 0.6 A. The X-ray analysis implies that protonation of normal flavin at N(1), as would occur in formation of the neutral fully reduced species, would result in a similar structural perturbation. The oxidation-reduction potentials of 1-deaza-FMN flavodoxin from M. elsdenii have been determined in the pH range 4.5-9.2. The oxidized/semiquinone equilibrium (E'0 = -160 mV at pH 7.0) displays a pH dependence of -60 mV per pH unit; the semiquinone/reduced equilibrium (E'0 = -400 mV at pH 7.0) displays a pH dependence of -60 mV per pH unit at low pH and is pH independent at high pH, with a redox-linked pK of 7.4. Spectral changes of fully reduced 1-deaza-FMN flavodoxin with pH suggest that this latter pK corresponds to protonation of the flavin ring system (the pK of free reduced 1-deaza-FMN is 5.6 [Spencer, R., Fisher, J., & Walsh, C. (1977) Biochemistry 16, 3586-3593]. The pK of reduced 1-deaza-FMN flavodoxin provides an estimate of the electrostatic interaction between the protein and the bound prosthetic group; the free energy of binding neutral reduced 1-deaza-FMN is more negative than that for binding the anionic reduced 1-deaza-FMN by 2.4 kcal.(ABSTRACT TRUNCATED AT 250 WORDS)

Clostridium↗
PubMed1988

Iron superoxide dismutase. Nucleotide sequence of the gene from Escherichia coli K12 and correlations with crystal structures.

The nucleotide sequence of the iron superoxide dismutase gene from Escherichia coli K12 has been determined. Analysis of the DNA sequence and mapping of the mRNA start reveal a unique promoter and a putative rho-independent terminator, and suggest that the Fe dismutase gene constitutes a monocistronic operon. The gene encodes a polypeptide product consisting of 192 amino acid residues with a calculated Mr of 21,111. The published N-terminal amino acid sequence of E. coli B Fe dismutase (Steinman, H. M., and Hill, R. L. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 3725-3729), along with the sequences of seven other peptides reported here, was located in the primary structure deduced from the K12 E. coli gene sequence. A new molecular model for iron dismutase from E. coli, based on the DNA sequence and x-ray data for the E. coli B enzyme at 3.1 A resolution, allows detailed comparison of the structure of the iron enzyme with manganese superoxide dismutase from Thermus thermophilus HB8. The structural similarities are more extensive than indicated by earlier studies and are particularly striking in the vicinity of the metal-ligand cluster, which is surrounded by conserved aromatic residues. The combined structural and sequence information now available for a series of Mn and Fe superoxide dismutases identifies variable regions in these otherwise very similar molecules; the principal variable site occurs in a surface region between the two long helices which dominate the N-terminal domain.

Amino Acid Sequence↗
PubMed1985

The structure of manganese superoxide dismutase from Thermus thermophilus HB8 at 2.4-A resolution.

An atomic model of tetrameric manganese superoxide dismutase from Thermus thermophilus HB8 has been built into an electron density map at 2.4 A resolution, using chemical sequences of Mn dismutases from Thermus aquaticus and Bacillus stearothermophilus. The monomer fold is structurally very similar to the fold of iron dismutase and comprises two domains, each contributing two ligands to the metal. The Mn(III) ion is bound by protein ligands assigned as His 28, His 83, Asp 165, and His 169. Near neighbors in the metal-ligand environment include a series of hydrophobic residues, Phe 86, Trp 87, Trp 131, and Trp 167. The hydroxyl groups of two Tyr residues, at 36 and 182, are less than 7 A from the metal, as is His 32. Gln 150 forms a bridge between Tyr 36 and Trp 131. These ligands and nearby residues are strongly conserved in the known sequences of Mn dismutases. Only one of the two oxygens of Asp 165 has been assigned as a metal ligand, so that in the current model four protein atoms bind Mn(III). These ligand atoms form part of an approximate trigonal bipyramid in which water may occupy an axial position on the side opposite His 28. The conformation of the protein is unusual in the vicinity of the first ligand, His 28, as a consequence of the insertion of an extra residue in an alpha-helix. The distortion of the helix allows His 32 to stack against the ligand, His 169, and brings Tyr 36 close to the Mn ion. Across one of the dimer interfaces, the two Mn ions are separated by about 18 A, and active center residues from adjoining subunits interdigitate; Tyr 172 interacts with His 32 of the neighboring chain and Glu 168 with the backbone of 168 and with the ligand His 169 from the opposite subunit. Only one other dimer interface occurs in the tetramer; it involves residues 55-62 and sequences near 140 and 156. The center of the oligomeric molecule is filled with solvent.

Amino Acid Sequence↗
PubMed1985

Crystallographic characterization of phthalate oxygenase reductase, an iron-sulfur flavoprotein from Pseudomonas cepacia.

Pseudomonads grown on phthalate synthesize a series of enzymes that metabolize this aromatic substrate. Among the inducible enzymes is a reductase which transfers electrons from NADH to the terminal dioxygenase that converts phthalate to the corresponding cis-1,2-dihydrodiol (Keyser, P. (1976) Ph. D. thesis, University of Miami; Ribbons, D. W., and Evans, W. C. (1960) Biochem. J. 76, 310-318). The phthalate oxygenase reductase induced in Pseudomonas cepacia is a single polypeptide chain (Mr approximately equal to 33,000) with two prosthetic groups, FMN and [2Fe-2S]. This oxidoreductase has been crystallized at pH 6.7 from polyethylene glycol 6000 in space group R3 with a = b = 113.4 A and c = 77.7 A (hexagonal indexing).

Crystallization↗
PubMed1984

Characterization of crystals of protocatechuate 3,4-dioxygenase from Pseudomonas cepacia.

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) from Pseudomonas cepacia (Mr = 198,000) has been crystallized from potassium phosphate at pH 7.5. Of the two orthorhombic forms that have been characterized, one contains a single molecule of tetrameric protocatechuate dioxygenase, (alpha beta Fe3+)4, per asymmetric unit. The space group and cell dimensions for this form are: P2(1)2(1)2(1) with a = 125.9 A, b = 161.7 A, and c = 85.2 A.

Azides↗
PubMed1984

Manganese and iron superoxide dismutases are structural homologs.

The crystal structure of a tetrameric manganese superoxide dismutase from a thermophilic bacterium, Thermus thermophilus HB8, has been determined at 4.4-A resolution by local averaging of electron density maps calculated by isomorphous replacement. The spatial arrangement of the principal secondary structural features of iron superoxide dismutase is conserved in manganese dismutase. The structural homology is displayed by orienting the polypeptide chain of Escherichia coli Fe dismutase in the electron density map of Mn dismutase. Densities corresponding to bound Mn3+ occur at locations equivalent to the Fe3+ positions in iron dismutase, indicating one metal binding site per chain, or four sites per tetramer. The Mn tetramer, with 222 symmetry, is approximately rectangular in shape and appears to be constructed with only two unique interfaces. One set of interchain contacts closely resembles the dimer interface of Fe dismutase, but the other interface utilizes an inserted polypeptide segment that has no equivalent in Fe dismutase.

Crystallization↗
PubMed1983

Studies of the ferredoxin from Thermus thermophilus.

The soluble ferredoxin from Thermus thermophilus was examined by Mössbauer and EPR spectroscopies and by reductive titrations. These studies demonstrate the presence of one 3Fe center, responsible for the characteristic g = 2.02 EPR signal in the oxidized protein, and one [4Fe-4S] center which is responsible for the rhombic EPR spectrum of the fully reduced protein. These assignments should replace those made by Ohnishi et al. (Ohnishi, T., Blum, H., Sato, S., Nakazawa, K., Hon-nami, K., and Oshima, T. (1980) J. Biol. Chem. 255, 345-348) prior to the discovery of the 3Fe clusters. The amino acid composition was determined and is discussed with reference to recent structural studies of 7Fe ferredoxins.

Amino Acids↗
PubMed1983

Structure of oxidized flavodoxin from Anacystis nidulans.

The structure of oxidized flavodoxin from the cyanobacterium Anacystis nidulans has been determined at 2.5 A resolution with phases calculated from ethylmercury phosphate and dimercuriacetate derivatives. The determination of partial sequences, including a total of 85 residues, has assisted in the interpretation of the electron density. Preliminary refinement of a partial model (1072 atoms) has reduced R to 0.349 for the 10.997 reflections between 2.0 and 5.0 A with 1 greater than 2 sigma. The polypeptide backbone, which comprises 167 residues in the current model, adopts the familiar beta-alpha-beta conformation found in other flavodoxins and in the nucleotide-binding domains of the pyridine-nucleotide dehydrogenases, with five parallel strands in the central sheet. Comparison with flavodoxin from Clostridium MP (138 residues) shows that extra residues of A. nidulans flavodoxin are accommodated in a major insertion about 20 residues in length, which forms a lobe adjacent to the fifth strand of parallel sheet, and in additions to several external segments. Residues added between the fourth sheet strand and the start of the third helix alter the environment of the pyrimidine end of the flavin mononucleotide ring. The flavin mononucleotide phosphate binds to the start of helix 1, interacting with hydroxyamino acids and with main-chain amide groups. Two hydrophobic residues, both tentatively identified as Trp, enclose the isoalloxazine ring; the solvent-exposed Trp is nearly parallel to the flavin ring. The hydrophobic environment provided by these residues must be partly responsible for the pronounced vibrational resolution of the flavin spectrum near 450 nm. The flavin ring is tilted relative to its orientation in Clostridium MP flavodoxin. In addition, atoms N-3 and O-2 alpha of the isoalloxazine appear to form hydrogen bonds to the backbone at CO97 and NH99 in a conformation entirely different from that found in Clostridium MP flavodoxin but structurally analogous to Desulfovibrio vulgaris flavodoxin.

Amino Acid Sequence↗
PubMed1983

Iron superoxide dismutase from Escherichia coli at 3.1-A resolution: a structure unlike that of copper/zinc protein at both monomer and dimer levels.

The structure of iron superoxide dismutase (EC 1.15.1.1) from Escherichia coli has been determined at 3.1-A resolution. The dimeric molecule is constructed from identical subunits, which are two-domain polypeptides. The NH2-terminal domain is composed of two antiparallel crossing helices and the COOH-terminal domain is a three-layered structure characterized by mixed alpha/beta secondary structural features. The active center iron atoms, separated by 18 A and located near the monomer-monomer interface, are coordinated by two amino acid residues from each domain. Azide binding has been investigated by using difference Fourier techniques. Consistent with the notion of the independent evolution of the copper/zinc dismutase gene, the iron dismutase structure resembles the copper/zinc protein at neither the monomer nor the dimer level.

Binding Sites↗
PubMed1981

Molecular complex of lumiflavin and 2-aminobenzoic acid: crystal structure, crystal spectra, and solution properties.

The molecular complex lumiflavin-2-aminobenzoic acid monohydrate (C13H12N4O2.C7H7NO2.H2O) crystallizes from from aqueous solution as red triclinic prisms. The space group is P1 with cell dimensions a = 9.660 A, b = 14.866 A, c = 7.045 A, alpha = 95.44 degrees , beta = 95.86 degrees, and gamma = 105.66 degrees . The crystal structure was solved by direct methods and refined by block-diagonal least-squares procedures to an R value of 0.050 on the basis of 1338 observed reflections. The structure is composed of stacks of alternating lumiflavin adn un-ionized (neutral) 2-aminobenzoic acid molecules. Two different modes of stacking interaction are observed. In one, 2-aminobenzoic acid overlaps all three of the isoalloxazine rings, at a mean distance of 3.36 A; in the other, 2-aminobenzoic acid interacts distance of 3.36 A; in the other, 2-aminobenzoic acid interacts with the pyrazine and dimethylbenzene moieties, at a distance of 3.42 A. Perpendicular to the stacking direction, the molecules form a continuous sheet. Each flavin is hydrogen bonded via O(2) and NH(3) to two symmetrically related aminobenzoates; the water of crystallization forms three hydrogen bonds, bridging two flavins, via O(4) and N(5), and one aminobenzoic acid. The red color of the crystals results from a charge-transfer transition involving stacked flavin and 2-aminobenzoic acid. The red color of the crystals results from a charge-transfer transition involving stacked flavin and 2-aminobenzoic acid molecules. Measurements of the polarized optical absorption spectra of crystals show that the transition moment direction for the long wavelength absorbance (beyond 530 nm) contains an out-of-plane component which can only arise from a charge-transfer interaction. Since the amino N does not make exceptionally close interactions with isoalloxazine atoms in either stacking mode (minimum interatomic distance 3.52 A), the charge transfer is presumed to involve pi orbitals of the 2-aminobenzoic acid donor.

Chemical Phenomena↗
PubMed1981

Characterization of crystals of tetrameric manganese superoxide dismutase from Thermus thermophilus HB8.

The tetrameric manganese superoxide dismutase from the extreme thermophile Thermus thermophilus HB8 crystallizes in space group P41212 (or its enantiomorph) with a = b = 147.5 A, c = 55.9 A. The diffraction patterns extent to 1.4 A, and the crystals are very resistant to decay induced by x-irradiation. Measurements of the crystal density in Ficoll gradients are consistent with an asymmetric unit containing the entire tetramer (Mr = 80,000).

Manganese↗
PubMed1975

Optical spectra and electronic structure of flavine mononucleotide in flavodoxin crystals.

The polarized single-crystal absorption spectra of the oxidized and semiquinone forms of flavodoxin from Clostridium MP have been measured with a double beam recording microspectrophotometer. The spectra establish that the radical species in the crystal is the neutral (blue) falvine semiquinone. Combination of the spectra reported here with polarization data from previous fluorescence and stretched-film studies provides transition moment directions for the first two phi-phi transitions of the oxidized form. Predictions of molecular orbital theory are in good agreement with these experimental directions. The crystal spectra of the semiquinone indicate that the two lowest frequency transitions have the same detailed orbital origin as the corresponding transitions of the oxidized form; in the semiquinone these transitions appear at lower frequency, are closer together, and, as predicted from detailed considerations of transition probabilities, exhibit approximately half the absorption intensity. Our hypothesis of a common orbital origin suggests that semiquinone formation takes place by the addition of an electron to the lowest empty phi orbital of the oxidized form without any gross electronic rearrangement.

Bacterial Proteins↗