Biomedical subjects

James P Allen

Publications and source records attributed to James P Allen.

9 recordsLinked to original sources

PubMed2006

Crystallization and structural analysis of GADPH from Spinacia oleracea in a new form.

Two crystalline forms of GADPH (D-glyceraldehyde-3-phosphate dehydrogenase) from Spinacia oleracea were obtained using sitting-drop vapor diffusion. Despite the very low concentration of GADPH in the solutions, two crystalline forms were obtained, one of which was the previously reported C222 space group with unit-cell parameters a = 155.3, b = 181.7, c = 107.6 A and the other of which belonged to a new space group I4(1)22, with unit-cell parameters a = b = 120.9, c = 154.5 A. Diffraction data were measured from both native and derivatives, yielding structures at a resolution limit of 3.0 A. Differences at the NAD(+)/NADP(+)-binding site seen in these structures compared with the previously reported structure with bound coenzyme suggest that conformational changes associated with pyridine-nucleotide binding may play a role in the regulation of this enzyme.

Binding Sites↗

PubMed2006

Structure of phenoxazinone synthase from Streptomyces antibioticus reveals a new type 2 copper center.

The multicopper oxidase phenoxazinone synthase (PHS) catalyzes the penultimate step in the biosynthesis of the antibiotic actinomycin D by Streptomyces antibioticus. PHS exists in two oligomeric forms: a dimeric form and a hexameric form, with older actinomycin-producing cultures containing predominately the hexameric form. The structure of hexameric PHS has been determined using X-ray diffraction to a resolution limit of 2.30 A and is found to contain several unexpected and distinctive features. The structure forms a hexameric ring that is centered on a pseudo 6-fold axis and has an outer diameter of 185 A with a large central cavity that has a diameter of 50 A. This hexameric structure is stabilized by a long loop connecting two domains; bound to this long loop is a fifth copper atom that is present as a type 2 copper. This copper atom is not present in any other multicopper oxidase, and its presence appears to stabilize the hexameric structure.

Copper↗

PubMed2005

Structure of spinach nitrite reductase: implications for multi-electron reactions by the iron-sulfur:siroheme cofactor.

The structure of nitrite reductase, a key enzyme in the process of nitrogen assimilation, has been determined using X-ray diffraction to a resolution limit of 2.8 A. The protein has a globular fold consisting of 3 alpha/beta domains with the siroheme-iron sulfur cofactor at the interface of the three domains. The Fe(4)S(4) cluster is coordinated by cysteines 441, 447, 482, and 486. The siroheme is located at a distance of 4.2 A from the cluster, and the central iron atom is coordinated to Cys 486. The siroheme is surrounded by several ionizable amino acid residues that facilitate the binding and subsequent reduction of nitrite. A model for the ferredoxin:nitrite reductase complex is proposed in which the binding of ferredoxin to a positively charged region of nitrite reductase results in elimination of exposure of the cofactors to the solvent. The structure of nitrite reductase shows a broad similarity to the hemoprotein subunit of sulfite reductase but has many significant differences in the backbone positions that could reflect sequence differences or could arise from alterations of the sulfite reductase structure that arise from the isolation of this subunit from the native complex. The implications of the nitrite reductase structure for understanding multi-electron processes are discussed in terms of differences in the protein environments of the cofactors.

Coenzymes↗

PubMed2005

Environmental control of primary photochemistry in a mutant bacterial reaction center.

The core structure of the photosynthetic reaction center is quasisymmetric with two potential pathways (called A and B) for transmembrane electron transfer. Both the pathway and products of light-induced charge separation depend on local electrostatic interactions and the nature of the excited states generated at early times in reaction centers isolated from Rhodobacter sphaeroides. Here transient absorbance measurements were recorded following specific excitation of the Q(y)() transitions of P (the special pair of bacteriochlorophylls), the monomer bacteriochlorophylls (B(A) and B(B)), or the bacteriopheophytins (H(A) and H(B)) as a function of both buffer pH and detergent in a reaction center mutant with the mutations L168 His to Glu and L170 Asn to Asp in the vicinity of P and B(B). At a low pH in any detergent, or at any pH in a nonionic detergent (Triton X-100), the photochemistry of this mutant is faster than, but similar to, wild type (i.e. electron transfer occurs along the A-side, 390 nm excitation is capable of producing short-lived B-side charge separation (B(B)(+)H(B)(-)) but no long-lived B(B)(+)H(B)(-) is observed). Certain buffering conditions result in the stabilization of the B-side charge separated state B(B)(+)H(B)(-), including high pH in the zwitterionic detergent LDAO, even following excitation with low energy photons (800 or 740 nm). The most striking result is that conditions giving rise to stable B-side charge separation result in a lack of A-side charge separation, even when P is directly excited. The mechanism that links B(B)(+)H(B)(-) stabilization to this change in the photochemistry of P in the mutant is not understood, but clearly these two processes are linked and highly sensitive to the local electrostatic environment produced by buffering conditions (pH and detergent).

Buffers↗

PubMed2004

Crystallization and initial X-ray analysis of phenoxazinone synthase from Streptomyces antibioticus.

Phenoxazinone synthase, an oligomeric multicopper oxidase produced by Streptomyces antibioticus, is responsible for the six-electron oxidative coupling of two molecules of 4-methyl 3-hydroxyanthraniloyl pentapeptide to form the phenoxazinone chromophore of the antineoplastic agent actinomycin D. Spectroscopic studies have shown that the enzyme contains one type I (blue) and three to four type II copper centers. However, the exact arrangement of the copper centers in this multicopper oxidase is unknown. As a first step towards determining the three-dimensional structure of the enzyme, phenoxazinone synthase has been crystallized. The hexameric form of phenoxazinone synthase was purified from 72 h cultures of S. lividans containing the plasmid pIJ702. Purified hexamers were concentrated to 75 mg ml(-1) and used to grow two forms of crystals. Data collected from the two crystal forms were processed in two separate space groups. Crystals of both forms were grown at 288 K using the sitting-drop vapour-diffusion method. Native data sets extending to resolutions of 3.35 and 2.30 A have been collected and processed in space groups R32 and P1, respectively.

Crystallization↗

PubMed2004

Crystallization of the mosquito-larvicidal binary toxin produced by Bacillus sphaericus.

The Bacillus sphaericus binary toxin is expressed during the early stages of sporulation and is composed of two separately encoded polypeptides: BinA (41.9 kDa) and BinB (51.4 kDa). The binary toxin forms microcrystalline inclusions inside the mother cell that, once ingested, are solubilized in the alkaline pH of the larval gut. B. sphaericus cultures were grown to complete sporulation, harvested and washed. The binary toxin was solubilized with 50 mM NaOH, purified using column chromatography and crystallized. Crystals were grown using the sitting-drop vapor-diffusion method in a glycine-buffered solution containing 0.5 M NaCl and 19.5% PEG 4000 as the precipitant. Native data were collected to a resolution of 2.7 A.

Bacterial Toxins↗

PubMed2004

My daily constitutional in martinsried.

The three-dimensional structures of bacterial reaction centers have served as the framework for much of our understanding of anoxygenic photosynthesis. A key step in the determination of the structure of the reaction center from Rhodobacter sphaeroides was the use the molecular replacement technique. For this technique, we made use of two sets of data. First, X-ray diffraction data had been measured from crystals of the reaction center from R. sphaeroides by our research group in California, led by George Feher and Douglas Rees. The second data set consisted of the coordinates of the three-dimensional structure of the reaction center from Rhodopseudomonas (now Blastochloris) viridis, which had been solved in the pioneering efforts of a group in Martinsried, led by Johann Deisenhofer, Robert Huber and Hartmut Michel. The collaborative efforts of these two groups to determine the structure of the reaction center from R. sphaeroides is described.

Journal Article↗

PubMed2004

Introduction.

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Journal Article↗

PubMed2003

The structure of the FMO protein from Chlorobium tepidum at 2.2 A resolution.

The bacteriochlorophyll protein, or FMO protein, from Chlorobium tepidum, which serves as a light-harvesting complex and directs light energy from the chlorosomes attached to the cell membrane to the reaction center has been crystallized in a new space group. The crystals belong to the cubic space group P4(3)32 and the structure has been refined to a resolution 2.2 A with a R factor of 19.7%. The electron density maps show that the structure is composed of two beta sheets that surround seven bacteriochlorophylls as previously reported (Li et al. (1997) J Mol Biol 271: 456-471). The availability of the new data allows a more accurate refinement of the pigment-protein complex including identification of bound solvent molecules. Several structural differences probably contribute to the observed spectroscopic differences between the FMO proteins from Cb. tepidum and Prosthecochloris aestuarii, including differences in the planarity of corresponding tetrapyrroles. A citrate molecule is found on the surface of each protein subunit of the trimer from Cb. tepidum. However, the citrate molecule is over 15 A from any bacteriochlorophyll. The presence of the citrate probably does not contribute to the function of the protein although it does contribute to the crystallization as it interacts with a crystallographically related trimer. Among the 236 water molecules found in the protein are four that appear to play a special role in the properties of bacteriochlorophyll 2, as this tetrapyrrole is coordinated by one of these water molecules and the waters form a hydrogen-bonded network that leads to the surface of the protein.

Journal Article↗