Neocarzinostatin chromophore: Presence of a cyclic carbonate subunit and its modification in the structure of other biologically active forms

On the basis of spectroscopic evidence, opening of a five-membered cyclic carbonate ring (1,3-dioxolan-2-one) in the C₁₅-subunit of the previously determined partial structure $\text{1}$ (Fig. 1) of the major neocarzinostatin chromophore (NCS-Chrom $\text{A}$), is proposed to account for its base-catalyzed methanolysis to NCS-Chrom $\text{C}$. NCS-Chrom $\text{B}$, apparently an authentic natural product present as a minor component in all preparations of NCS studied, was found to be formally equivalent to the hydrolysis/decarboxylation product of the cyclic carbonate functionality in NCS-Chrom $\text{A}$. The mercaptan-dependent DNA strand-scission activity, equivalent for NCS-Chrom $\text{A}$, $\text{B}$ and $\text{C}$, is independent of the integrity of the cyclic carbonate ring system and implicates a secondary site in the C₁₅-substructure for mercaptan activation.     

Solution conformation and crystal structure of methyl 3,6-dideoxy-β-d-ribohexopyranoside,an immunodominant sugar of O-antigens

The crystal structure of methyl 3,6-dideoxy-β-d-ribohexopyranoside monohydrate was determined by direct methods. Crystals are monoclinic, space group P2₁, with cell dimensions $\text{a = 9.089(1), b = 7.668(1), c = 6.956(1)}\text{A}\text{?}\text{, β = 101.12°}$. The molecule adopts the ⁴C₁ chair conformation. The same conformation was also found in both aqueous and chloroform solutions. The pyranose ring is only slightly distorted, and the consequences of this observation on antigen structure are discussed.     

Crystallizations and preliminary X-ray studies of calotropins DI and DII

Crystals of calotropin DI (M_r 23,400), have been prepared by microdialysis against 5% (w/v) polyethylene glycol 20,000 in water, pH 7.0. They have orthorhombic space group P2₁2₁2₁ with cell parameters $\text{a = 57.5}\text{A}\text{?}\text{, b = 86.2}\text{A}\text{?}\text{, c = 40.3}\text{A}\text{?}$. Crystals of calotropin DII (M_r 24,000), prepared by the same technique against 5% (w/v) polyethylene glycol 20,000 in phosphate buffer of low ionic strength, pH 7.0, display monoclinic space group C2 with cell parameters $\text{a = 135.8}\text{A}\text{?}\text{, b = 32.0}\text{A}\text{?}\text{, c = 47.7}\text{A}\text{?}\text{, β = 103.80 °}$. In both cases, there is only one molecule in the asymmetric unit.     

Leukotriene A4: Enzymatic conversion to leukotriene C4

An unstable epoxide, leukotriene A₄ (5(S)-$\text{trans}$-5,6-oxido-7,9-$\text{trans}$-11,14-$\text{cis}$-eicosatetraenoic acid), was earlier proposed to be an intermediate in the conversion of arachidonic acid into the slow reacting substance (SRS), leukotriene C₄. In the present work synthetic leukotriene A₄ was incubated with human leukocytes or murine mastocytoma cells. A lipoxygenase inhibitor, BW755C, was added in order to prevent leukotriene formation from endogenous substrate. Leukotriene C₄ and 11-$\text{trans}$-leukotriene C₄ were the main products with SRS activity. It was not established whether the 11-$\text{trans}$-compound was formed by isomerization at the leukotriene A₄ or C₄ stage.     

Crystallization and preliminary X-ray diffraction studies of colicin E3 immunity protein

Immunity protein, an inhibitor of the ribonuclease activity of the protein antibiotic colicin E₃, crystallizes in the orthorhombic space group C222 with cell dimensions $\text{a = 78·7}\text{A}\text{?}\text{, b = 54·1}\text{A}\text{?}\text{, c = 36·1}\text{A}\text{?}$ and one molecule of M_r 9800 per asymmetric unit. The crystals are suitable for high resolution X-ray analysis.     

Molecular conformation of prostaglandin A1

The crystal and molecular structure of prostaglandin A₁ (PGA₁) has been determined by X-ray diffraction. (Space group P2₁2₁2₁, $\text{a}\text{= 18.10}\text{A}\text{?}\text{A}$, $\text{b}\text{= 21.09}\text{A}\text{?}\text{A}\text{,}\text{c}\text{= 5.42,}\text{Z}\text{= 4)}$. Comparison of the structure of PGA₁ with that of PGF_1β (determined previously as the tribromobenzoate) indicates significant differences in the relative intramolecular side chain orientations. However, conformation and torsional angles within each side chain of PGA₁ are retained with little or no change in comparison with PGF_1β, indicating the presence of the C₁₅-bromobenzoate groups in the latter have had little effect in altering internal side chain conformation in the solid state. The overall differences in relative side chain orientation in PGA₁ are attributable to chemical and conformational changes in the substituted cyclopentane moiety (C₈-C₁₂).     

Crystallization of a DNA-unwinding protein: Preliminary X-ray analysis of fd bacteriophage gene 5 product

The gene 5 protein from bacteriophage fd, which binds to single-stranded progeny fd DNA, was obtained as large single crystals and subjected to X-ray diffraction analysis. The crystals are of monoclinic space group C2 with $\text{a = 75.8}\text{A}\text{?}\text{, b = 28.0}\text{A}\text{?}\text{, c = 42.5}\text{A}\text{?}\text{and}\text{β = 103 °12′}$. The unit cell has one molecule of 9800 daltons as the asymmetric unit.     

Manganese superoxide dismutases from Escherichia coli and from yeast mitochondria: Preliminary X-ray crystallographic studies

The Mn superoxide dismutase from Escherichia coli has been obtained in three crystal forms: (I) from 68% saturated (NH₄)₂SO₄, space group P222 or P222₁, $\text{a = 47}\text{A}\text{?}\text{, b = 103}\text{A}\text{?}\text{, c = 47.5}\text{A}\text{?}$, with one subunit per asymmetric unit; (II) from 50% polyethylene glycol 6000, space group C222₁ (with approx. P4₁2₁2 symmetry), $\text{a = 101}\text{A}\text{?}\text{, b = 108}\text{A}\text{?}\text{, c = 180}\text{A}\text{?}$, with four subunits (2 molecules) per asymmetric unit; (III) from 52% polyethylene glycol with a different method of preparing the enzyme solution, space group P2₁2₁2, $\text{a = 47}\text{A}\text{?}\text{, b = 51}\text{A}\text{?}\text{, c = 188}\text{A}\text{?}$, with two subunits per asymmetric unit.The yeast mitochondrial Mn superoxide dismutase has yielded the same crystal form both from 30% 2-methyl-2,4-pentane diol and from 23% polyethylene glycol 6000: space group P2₁2₁2₁, $\text{a = 63}\text{A}\text{?}\text{, b = 115}\text{A}\text{?}\text{, c = 125}\text{A}\text{?}$, with four subunits (one molecule) per asymmetric unit.A full X-ray crystallographic study of at least one of these enzymes is planned.     

Crystallization of and preliminary X-ray diffraction data for TET-repressor and the TET-repressor-tetracycline complex

The TET-repressor encoded by the transposon Tn10 has been crystallized along with the repressor-tetracycline complex. Both crystals belong to the space group P4₃2₁2 (or P4₁2₁2) with cell dimensions $\text{a = b = 74.3(1)}\text{A}\text{?}\text{, c = 94.2(2)}\text{A}\text{?}\text{and}\text{a = b = 73.3(1)}\text{A}\text{?}\text{, c = 94.6(2)}\text{A}\text{?}$ for the free and complexed repressor, respectively. There is one molecule of molecular weight 23,000 per asymmetric unit, and the biologically active dimer therefore consists of two identically formed subunits which are related by a crystallographic 2-fold axis. This isomorphism of TET-repressor and its tetracycline complex suggests that only minor, subtle changes in structure trigger binding to or release of the operator. The crystals of the native protein permit X-ray data collection to 3.2 Å and those of the complexed repressor to 2.8 Å.     

Crystallographic analysis of the phytoagglutinin from Abrus precatorius by X-ray diffraction and electron microscopy

The lectin from the seeds of Abrus precatorius has been crystallized and the crystals subjected to study by X-ray diffraction and electron microscopy. Three closely related crystal forms were obtained, of orthorhombic space group P2₁2₁2₁ with $\text{a = 138}\text{A}\text{?}$, $\text{b = 142}\text{A}\text{?}$, and $\text{c = 173}\text{A}\text{?}$, of tetragonal space group P4₁2₁2 with $\text{a = b = 136}\text{A}\text{?}$, $\text{c = 176}\text{A}\text{?}$, and a twinned intermediate of the first two. From electron microscopy and two-dimensional spatial filtering of electron micrographs of the crystals, the molecule appears to consist of four similar domains grouped in a roughly planar diamond-shaped arrangement having a local intramolecular dyad axis. The average diameter of the Abrus lectin molecule is 50 to 60 Å and the individual domains appear to have a diameter of about 25 Å.     

Crystal data, molecular dimensions and molecular symmetry in cytochrome oxidase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa cytochrome oxidase (nitrite reductase, cytochrome cd) has been crystallized in space group P2₁2₁2 with cell dimensions $\text{a = 122.8}\text{A}\text{?}\text{, b = 87.2}\text{A}\text{?}\text{, c = 73.4}\text{A}\text{?}$. Density measurements suggest that the asymmetric unit contains one 63,000 molecular weight subunit of the dimeric molecule. Crystal data agree well with electron microscopy of single molecules. The X-ray pattern extends beyond 2.5 Å resolution, and structure analysis is in progress.     

Synthesis of 3,4-13C2 steroids

A-ring enollactones $\text{1a}$, $\text{1b}$ or $\text{9}$ derived from 4-cholesten-3-one, testosterone benzoate or 3-oxo-4-estren-17β-yl benzoate were condensed with [1,2-¹³C₂]acetyl chloride to give intermediates $\text{2a}$, $\text{2b}$ or $\text{10}$. $\text{2a}$ and $\text{2b}$ were cyclized by acid or base to give 3,4-¹³C₂-labeled 4-cholesten-3-one and testosterone, respectively. [3,4-¹³C₂]4-Cholesten-3-one was converted via reduction of its trimethylsilyl enol ether to [3,4-¹³C₂]cholesterol. Acetyl enollactone $\text{10}$ was cyclized in acetic acid to [3,4-¹³C₂]3-oxo-4-estren-17β-yl benzoate followed by aromatization and hydrolysis to produce [3,4-¹³C₂]estradiol-17β. Alternatively, cyclization of $\text{10}$ with base afforded [3,4-¹³C₂]3-oxo-4-estren-17β-ol directly, which was then oxidized and aromatized to yield [3,4-¹³C₂]estrone. Ozonolysis of progesterone, conversion to the diketal ester $\text{16}$ and acylation followed by acid hydrolysis furnished [3,4-¹³C₂]progesterone.     

A cobalt porphyrin containing protein reducible by hydrogenase isolated from Desulfovibrio desulfuricans (Norway)

A cobalt-porphyrin containing protein has been isolated from the sulfate-reducer $\text{Desulfovibrio desulfuricans}$ (Norway). This violet-colored protein has a molecular weight of approx. 13,000 daltons and contains 1 cobalt atom/molecule. The apo-protein was estimated to contain 104 amino-acid residues giving a molecular weight of 11,000 daltons. The UV-visible absorption spectrum of the protein exhibiting maxima at 588,418 and 280 nm with a shoulder at 550 nm is characteristic of metalloporphyrin proteins. The molar extinction coefficients of the cobalt-protein at 588, 418 and 280 nm are 31,330 , 64,670 and 17,200 respectively and its absorbance ratio $\text{A}{}_{280}\text{A}{}_{588}$ is 0.54. The protein is reduced by dithionite giving a blue-colored reduced form. Important spectral modifications of the chromophore occurred during the reduction including a shift of the Soret peak from 418 to 381 nm and a shift of the α band in the opposite direction from 588 to 593.5 nm. The Co-protein was slowly reduced by the hydrogenase from $\text{D.desulfuricans}$ under hydrogen in the presence of cytochrome C₃. The reported data suggest that the redox states of the cobalt center of this new electron carrier correspond to the Co(III) and Co(II) states.     

Corrections to Nomenclature of Phosphorus-Containing Compounds of Biochemical Importance

Crystals of cholesteryl oleate (C₄₅H₇₈O₂) are monoclinic, space group P2₁, with $\text{a = 12.65(3), b = 9.13(3), c = 18.79(5)}\text{A}\text{?}\text{, β = 93.3(3)°}$ and have 2 molecules in the unit cell. The crystal structure has been determined by Patterson and Fourier methods at a resolution $\text{d}{}_{\mathrm{<mtext>min</mtext>}}\text{= 1.1}\text{A}\text{?}$, using 799 X-ray intensities (CuKα) measured by a diffractometer. Structure refinement by block-diagonal least squares gave R = 0.12. The oleate chains are almost straight except for a kink at the cis-double bond. The chains pack side by side but without a regular sub-cell structure, in a manner which might be similar to the arrangement within biological membranes. As in cholesteryl octanoate, the cholesteryl ring systems pack together with extensive overlap of anti-parallel nearest neighbours. Projecting methyl groups interlock.     

Crystallization and preliminary crystallographic data for cytochrome c551.5 (c7) from Desulfuromonas acetoxidans.

Cytochrome c₇, a low potential, three heme-containing protein (molecular weight 9800) was isolated from Desulfuromonas acetoxidans and crystallized from polyethylene glycol solutions. The crystals belong to the orthorhombic space group P2₁2₁2₁, with unit-cell dimensions $\text{a = 44·10}\text{A}\text{?}\text{, b = 37·55}\text{A}\text{?}$ and $\text{c = 45·08}\text{A}\text{?}$ and have one molecule of protein per asymmetric unit.     

Gibberellin biosynthesis in a cell-free system from immature seeds of Pisum sativum

A cell-free system from immature pea seeds converts ¹⁴C-labelled $\text{ent}$-kaurene to $\text{ent}$-kaurenol, $\text{ent}$-kaurenal, $\text{ent}$-kaurenoic acid, $\text{ent}$-7α-hydroxykaurenoic acid, and gibberellin A₁₂-aldehyde. The latter becomes converted further to 13-hydroxygibberellin A₁₂, gibberellin A44, gibberellin A₁₂-alcohol, and several unidentified products. Thus the biosynthesis of gibberellins $\text{via ent}$-kaurene is now established for a member of the $\text{Leguminosae}$. It is the first time that 13-hydroxylation of gibberellins has been observed in a cell-free system and that gibberellin A₁₂-alcohol has been obtained in any biological system.     

Crystallographic data of the selenoenzyme glutathione peroxidase

Glutathione peroxidase prepared from bovine erythrocytes yields small, but well-ordered plate-like crystals. X-ray investigation shows them to belong to monoclinic space group C2. Unit cell dimensions are: $\text{a = 90.4}\text{A}\text{?}\text{, b = 109.5}\text{A}\text{?}\text{, c = 58.6}\text{A}\text{?}$, β = 99 ° ± 15 min. The crystal density is ?_c = 1.36 ± 0.02 g.cm^?3. Consequently, the asymmetric unit of the crystal cell is occupied by one tetrameric molecule of M_r 84,000. Matthew's (1968) parameter Γ_M is calculated to be 1.71 ų/dalton.     

Preliminary X-ray studies on Pseudomonas isoamylase

Single crystals of Pseudomonas isoamylase (M_r 95,000) belong to space group P2₁2₁2₁ with unit cell dimensions of $\text{a = 137.9}\text{A}\text{?}\text{, b = 52.9}\text{A}\text{?}\text{, c = 151.2}\text{A}\text{?}$. A Guinier plot of the X-ray small-angle scattering of the protein solution gave the radius of gyration of the molecule as 27.5 Å.     

Purification and preliminary crystallographic studies on azurin and cytochrome c' from Alcaligenes denitrificans and Alcaligenes sp. NCIB 11015.

Purification and crystallisation procedures are reported for azurin and cytochrome c′ from Alcaligenes denitrificans and Alcaligenes sp. NCIB 11015. The azurin crystals from A. denitrificans are suitable for high-resolution X-ray structure analysis. They are orthorhombic, space group C222₁ (with marked tetragonal pseudo-symmetry), cell dimensions $\text{a = 75.0}\text{A}\text{?}\text{, b = 74.1}\text{A}\text{?}\text{, c = 99.5}\text{A}\text{?}$, with two molecules per asymmetric unit. The cytochrome c′ crystals from both species are hexagonal, space group P6₁22 (or P6₅22), cell dimensions $\text{a = b = 54.7}\text{A}\text{?}\text{, c ~ 185}\text{A}\text{?}$, γ = 120 °, with one subunit (molecular weight 14,000) in the asymmetric unit.     

Preliminary crystallographic data for lysozyme produced by Streptomyces erythraeus.

Crystals of lysozyme from Streptomyces erythraeus are orthorhombic, space group P2₁2₁2₁ with unit cell dimensions: $\text{a = 50.01}\text{A}\text{?}\text{, b = 61.98}\text{A}\text{?}$ and $\text{c = 67.85}\text{A}\text{?}$; and one molecule, of molecular weight about 18,500, per asymmetric unit.