Chemistry of the “molecular trap” of protease-catalyzed splicing reaction of complementary segments of α-subunit of hemoglobin A

The complementary fragments of human Hb α, α_1–30, and α_31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction. The segments α_24–30 and α_31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) α_24–40 has been manipulated by engineering the amino acid replacements to the positions α²⁷ and α³¹ to delineate the structural basis of the molecular trap. The location of Glu²⁷ and Arg³¹ residues in the contiguous segment α_24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at α²⁷ and α³¹ facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu³⁰-Arg³¹ peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.     

New catalyst design for polymerization of norbornene esters by reducing intramolecular interaction

We have used density functional theory to study palladium-based catalysts commonly used for the polymerization of norbornene derivatives with an ester group. Exo–exo, exo–endo, and endo–endo isomers of catalyst complexes were investigated; the endo–endo isomer was the most stable and inactive due to an intramolecular interaction between Pd and O of the carbonyl group. Phosphine groups are effective in minimizing the Pd–O interaction in the endo–endo isomer and P(C₆H₁₁)₃ was found to be the most efficient reagent. The intramolecular Pd–O interactions were estimated using model complexes, and it was demonstrated that they play a crucial role in stabilizing the endo–endo isomer.Figure Methyl ester norbornene complex for endo–endo isomers with P(C₆H₁₁)₃     

Deuterium labeling of norbornene derivatives by iridium catalyzed H/D exchange catalysis

The H/D exchange catalysis using the Ir(I) complex [Tp^Me₂Ir(η⁴-2,3-dimethylbutadiene)] (Tp^Me₂=hydridotris(3,5-dimethylpyrazolyl)borate) as the precatalyst was studied for selective deuteration of norbornene derivatives. In dependence of the norbornene substitution in 2,3 positions, selective deuteration of the norbornene double bond could be achieved. (±)-endo,exo-6-Deutero-bicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid diethyl ester was isolated in 82% yield.     

The use of norbornene derivatives in the synthesis of conformationally constrained peptides and pseudo-peptides

Summary The desymmetrisation ofendo-norborn-5-ene-2,3-dicarboxylic anhydride by proline esters has been used to prepare conformationally constrained pseudo-peptides with two peptide chains parallel to one another. A Curtius rearrangement on the desymmetrication adduct produced the corresponding isocyanate which was used to prepare both a peptide incorporating anendo-2-amino-3-carboxy-norborn-5-ene unit, and a pseudo-peptide with two peptide chains parallel to one another but offset by the presence of a urea unit. The conformational analysis of the resulting peptides was carried out, and the norbornene unit was found to induce the formation of β-turns and parallel β-sheets.     

Novel Nociceptin Analogues: Synthesis and Biological Activity

Syntheses are described of the nociceptin (1–13) amide [NC(1–13)-NH₂] and of several analogues in which either one or both the phenylalanine residues (positions 1 and 4), the arginine residues (positions 8 and 12) and the alanine residues (positions 7 and 11) have been replaced by N-benzyl-glycine, N-(3-guanidino-propyl)-glycine and β-alanine, respectively. The preparation is also described of NC(1–13)-NH₂ analogues in which either galactose or N-acetyl-galactosamine are β-O-glycosidically linked to Thr⁵ and/or to Ser¹⁰. Preliminary pharmacological experiments on mouse vas deferens preparations showed that Phe⁴, Thr⁵, Ala⁷ and Arg⁸ are crucial residues for OP₄ receptor activation. Manipulation of Phe¹ yielded peptides endowed with antagonist activity but [Nphe¹] NC(1–13)-NH₂ acted as an antagonist still possessing weak agonist activity. Introduction of the βAla residue either in position 7 or 11 of the [Nphe¹] NC(1–13)-NH₂ sequence, abolished any residual agonist activity and [Nphe¹, βAla⁷] NC(1–13)-NH₂ and [Nphe¹, βAla¹¹] NC(1–13)-NH₂ acted as competitive antagonists only. Modification of both Ala⁷ and Ala¹¹ abolished the antagonist activity of [Nphe¹]NC(1–13)-NH₂ probably by hindering receptor binding. Changes at positions 10 and 11 gave analogues still possessing agonist activity. [Ser(βGal)¹⁰] NC(1–13)-NH₂ displayed an activity comparable with that of NC(1–13)-NH₂, [Ser(βGalNAc)¹⁰] NC(1–13)-NH₂ and [βAla¹¹] NC(1–13)-NH₂ were five and 10 times less active, respectively.The α-amino acid residues are of the l-configuration. Standard abbreviations for amino acid derivatives and peptides are according to the suggestions of the IUPAC-IUB Commission on Biochemical Nomeclature (1984), Eur. J. Biochem. 138, 9–37. Abbreviations listed in the guide published in (2003), J. Peptide Sci. 9, 1–8 are used without explanation.     

<Emphasis Type="Italic">Ab initio</Emphasis> and DFT investigation of electrophilic addition reaction of bromine to <Emphasis Type="Italic">endo,endo</Emphasis>-tetracyclo[4.2.1.1<Superscript>3,6</Superscript>.0<Superscript>2,7</Superscript>]dodeca-4,9-diene

Full geometric optimization of endo,endo-tetracyclo[4.2.1.1^3,6.0^2,7]dodeca-4,9-diene (TTDD) has been carried out by ab initio and DFT/B3LYP methods and the structure of the molecule investigated. The double bonds of TTDD molecule are endo pyramidalized. The structure of π-orbitals and their mutual interactions for TTDD molecule were investigated. The cationic intermediates and products obtained as a result of the addition reaction have been studied using the HF/6-311G(d), HF/6-311G(d,p) and B3LYP/6-311G(d) methods. The bridged bromonium cation isomerized into the more stable N- and U-type cations and the difference between the stability of these cations is small. The N- and U-type reaction products are obtained as a result of the reaction, which takes place via the cations in question. The stability of exo, exo and exo, endo isomers of N-type product are nearly the same and the formation of both isomers is feasible. The U-type product basically formed from the exo, exo-isomer. Although the U-type cation was 0.68 kcal mol⁻¹ more stable than the N-type cation, the U-type product was 4.79 kcal mol⁻¹ less stable than the N-type product. Figure The energy diagram of TTDD–Br₂ system (kcal mol⁻¹)(MP2/6-311G*//HF/6-311G*)     

The Interfacial Tension of the Lipid Membrane Formed from Lipid–Amino Acid Systems

The interfacial tension of lipid membranes composed of phosphatidylcholine (lecithin, PC)–valine (Val), phosphatidylcholine–isoleucine (Ile), phosphatidylcholine–tyrosine (Tyr), and phosphatidylcholine–phenylalanine (Phe) has been studied. The membrane components formed 1:1 complexes. The interfacial tension measurements were used to determine the membrane surface concentration A ₃⁻¹, the membrane interfacial tension γ₃, and the stability constant K.     

Molecular and crystal structure of Nα-(9-fluorenyl)- methoxycarbonyl-L-ornithine hydrochloride diethyl ether solvate

Summary The solid-state conformation of the first N-protected ornithine derivative has been established by X-ray analysis. The hydrochloride of N^α-(9-fluorenyl)methoxycarbonyl-l-ornithine crystallises as diethyl ether solvate. The backbone (ω₀ ϕ, ψ χ¹) torsion angles are (174.9°,–84.0°, 145.9°,–171.0°). The conformation of the urethane amide bonds istrans. The ornithine aliphatic side chain adopts preferred fully extended conformation which is stabilised by the hydrogen bonding of the-NH ₃ ⁺ group to the diethyl ether molecule, carboxyl group and Cl⁻ anions.     

Magnesium complexes of the antiviral 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) and of its 1-, 3-, and 7-deaza analogues in aqueous solution

 The stability constants of the 1 : 1 complexes formed between Mg²⁺ and the anions of the N1, N3, and N7 deaza derivatives of 9-[2-(phosphonomethoxy)ethyl]adenine (PA^2–), i.e., of Mg(H;PA)⁺ and Mg(PA), were determined by potentiometric pH titration in aqueous solution (25  °C; I=0.1 M, NaNO₃) and compared with previous results [Sigel H, et al. (1992) Helv Chim Acta 75 : 2634–2656], obtained under the same conditions, for the corresponding complexes of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA^2–) and (phosphonomethoxy)ethane (PME^2–). Based on the analysis of a microconstant scheme it is concluded that in the monoprotonated complexes, Mg(H;PA)⁺, Mg²⁺ is coordinated to a significant part at the nucleobase, H⁺ being at the phosphonate group. By making use of log K ^Mg _Mg(R-PO3) versus pK ^H _H(R-PO3) straight-line plots (also obtained previously; see above) for simple phosphonates and phosphate monoesters, it is shown that all the Mg(PA) complexes, including those with PMEA^2– and PME^2–, are more stable than expected on the basis of the basicity of the ―PO^2– ₃ group. This proves that, to some extent, five-membered chelates, Mg(PA)_cl/O, involving the ether oxygen of the ―CH₂―O―CH₂―PO^2– ₃ chain are formed; their formation degree amounts to about 30–40% in equilibrium with the isomer having only a phosphonate-Mg²⁺ coordination. In the case of Mg(1-deaza-PMEA), probably a further isomer occurs in which also N3 of the nucleobase participates. The different properties between the Mg(PA) species and the Mg(AMP) complex are discussed. Received: 26 January 1998 / Accepted: 19 May 1998     

Ab initio and DFT study on the electrophilic addition reaction of bromine to tetracyclo[5.3.0.02,6.03,10]deca–4,8–diene

The electronic and geometric structures of tetracyclo[5.3.0.0^2,6.0^3,10]deca-4,8-diene (hypostrophene) have been investigated by ab initio and DFT/B3LYP methods using the 6-31G* and 6-311G* basis sets. The double bonds of hypostrophene are endo-pyramidalized. The cationic intermediates and products formed in the addition reaction have been investigated using the HF/6-311G*, HF/6-311G**, and B3LYP/6-311G* methods. The bridged bromonium cation was more stable than the U-type cation. Considering that the bridged cation does not isomerize to the less stable U-type cation, it is not possible for the U-type product to be obtained in the reaction. The bridged bromonium cation transformed into the more stable N-type cation and the N-type product was obtained via this cation. The thermodynamic stability of the exo, exo and exo, endo isomers of the N-type dibromide molecule were almost identical. The N-type product was 16.6 kcal mol⁻¹ more stable than the U-type product. Figure General energy diagram of the hypostrophene–bromine (HS–Br₂) system (kcal mol⁻¹) (MP2/6-311G*//HF/6-311G*)     

Expression of HLA class I molecules assembled with structural variants of human β2-microglobulin

 Mouse and human β₂-microglobulin (β₂m), which differ by 30% in their primary sequence, give rise to disparate levels of HLA class I heavy chain expression in transfectants of the β₂m-null FO-1 human melanoma cell line, i.e., mouse β₂m directs expression of HLA class I heavy chains that is only ∼20%–30% of that observed for heavy chains assembled with human β₂m. In this report we describe our efforts to better understand the structural basis of this regulatory phenomenon. Initial insight into the importance of the N-terminus of β₂m on HLA expression came from studies with FO-1 cells transfected with chimeric (human X mouse) B2m genes. Chimeric β₂m molecules containing residues 1–69 from human β₂m and residues 70–99 from mouse β₂m (designated HM- β₂m) induced expression of HLA heavy chains to a significantly greater extent (∼80% of level observed with cognate β₂m) than the reverse chimeric construct (designated MH- β₂m) (10%–15% of level observed with cognate β₂m). These data are consistent with the view that the major determinants of HLA class I heavy chain expression map to the portion of the β₂m molecule which forms the four-stranded β-pleated sheet, comprised of S1, S2, S4, and S5, and one strand of the three-stranded sheet (S3). The mapping of class I regulatory sites to the portion of β₂m containing the four-stranded β-pleated sheet supports the interpretation that the heavy chain contact residues on β₂m play the major role in regulating major histocompatibility (MHC) class I expression. To further dissect β₂m-mediated regulation of HLA class I expression, site-directed mutants of β₂m were prepared by conversion of human β₂m to the mouse sequence at individual amino acid positions within the four-stranded and three-stranded β-pleated sheets. Human to mouse amino acid substitutions were made in each divergent residue between positions 1–66, and as controls for COOH-terminal modification, several residues between positions 75 and 94. Cytofluorometry with HLA class I-specific antibodies indicated that cell surface expression of HLA class I heavy chains was largely insensitive to each of the individual substitutions. It is concluded that a combination of divergent residues mapping to positions of heavy chain contact are responsible for the differences observed in MHC class I expression by heterologous forms of β₂m. Received: 18 March 1997 / Revision: 21 April 1997     

The alpha-helix,an overlooked molecular motor

Summary. At first sight the alpha-helix appears as a rigid scaffold braced by hydrogen bonds nearly parallel to the helix axis. Looked at more closely it turned out to be highly dynamic and able to transform chemical into mechanical energy. The hydrogen bonds are fairly weak and compliant bonds. Their length, usually between 0.267 and 0.291 nm (mean value, 0.28 nm), depends on the interaction of the side chains. The most important strong interaction is the electrostatic repelling force between equally charged side chains (Glu⁻, Asp⁻, Lys⁺, Arg⁺), well known by experiments with polyamino acids. In proteins with different amino acids, repelling forces between charged side chains work in the axial direction and stretch the hydrogen bonds. Extreme shortening of the hydrogen bonds occurs when ions, e.g., Ca²⁺, H⁺, or PO₃⁻, are added and discharge side chains. This means a cooperative pitch decrease of the alpha-helix (pitch range between 0.52 and more than 0.55 nm; mean value, 0.54 nm). This pitch change is absolutely connected by steric reasons with torque generation and torsional rotations, as demonstrated by molecular and tubular alpha-helix models. Thus, charged alpha-helices are molecular motors propelled by the electrostatic energy of added ions. The motor effect is most striking with highly charged alpha-helical coiled coils, e.g., tropomyosin, myosin, and alpha-actinin that can rotate actin filaments by winding and unwinding. For example, the shortening of muscle depends on the sliding (drilling) motion of the Ca²⁺-activated helical actin filaments into the cross-bridges of the A-band. Here, models are presented for the in vitro sliding of actin filaments and for cytoplasmic streaming by winding and unwinding of myosin chains, and for membrane proteins that contain nonhelical domains between membrane-penetrating alpha-helices. They may transport molecules by the described torsional rotations if they perform supercoiling. Winding and supercoiling can lead to displacement of bound ions and to a feed-back-regulated oscillation between two different coiling stages E₁ and E₂ that explain “eversion”. The models need the torque for 1–2 rotations. They explain active and passive transports, the driving-effects of ion gradients, ATP hydrolysis by unwinding, ATP synthesis by winding up of the supercoils, etc. Correspondence and reprints: Linzerstrasse 72, 4810 Gmunden, Austria.     

Studies of H4R antagonists using 3D-QSAR,molecular docking and molecular dynamics

Three-dimensional quantitative structure–activity relationship studies were performed on a series of 88 histamine receptor 4 (H4R) antagonists in an attempt to elucidate the 3D structural features required for activity. Several in silico modeling approaches, including comparative molecular field analysis (CoMFA), comparative similarity indices analysis (CoMSIA), molecular docking, and molecular dynamics (MD), were carried out. The results show that both the ligand-based CoMFA model (Q ² = 0.548, R _ncv² = 0.870, R _pre² = 0.879, SEE = 0.410, SEP = 0.386) and the CoMSIA model (Q ² = 0.526, R _ncv² =0.866, R _pre² = 0.848, SEE = 0.416, SEP = 0.413) are acceptable, as they show good predictive capabilities. Furthermore, a combined analysis incorporating CoMFA, CoMSIA contour maps and MD results shows that (1) compounds with bulky or hydrophobic substituents at positions 4–6 in ring A (R2 substituent), positively charged or hydrogen-bonding (HB) donor groups in the R1 substituent, and hydrophilic or HB acceptor groups in ring C show enhanced biological activities, and (2) the key amino acids in the binding pocket are TRP67, LEU71, ASP94, TYR95, PHE263 and GLN266. To our best knowledge, this work is the first to report the 3D-QSAR modeling of these H4R antagonists. The conclusions of this work may lead to a better understanding of the mechanism of antagonism and aid in the design of new, more potent H4R antagonists.     

Disulfide Linkages and a Three-Dimensional Structure Model of the Extracellular Ligand-binding Domain of Guanylyl Cyclase C

Guanylyl cyclase C (GC-C) is a single-transmembrane receptor that is specifically activated by endogenous ligands, including guanylin, and the exogenous ligand, heat-stable enterotoxin. Using combined HPLC separation and MS analysis techniques the positions of the disulfide linkages in the extracellular ligand-binding domain (ECD) of GC-C were determined to be between Cys⁷–Cys⁹⁴, Cys⁷²–Cys⁷⁷, Cys¹⁰¹–Cys¹²⁸ and Cys¹⁷⁹–Cys²²⁶. Furthermore, a three-dimensional structural model of the ECD was constructed by homology modeling, using the structure of the ECD of GC-A as a template (van den Akker et al., 2000, Nature, 406: 101–104) and the information of the disulfide linkages. Although the GC-C model was similar to the known structure of GC-A, importantly its ligand-binding site appears to be located on the quite different region from that in GC-A.     

Probing the ground state of the purple mixed valence CuA center in nitrous oxide reductase: a CW ENDOR (X-band) study of the 65Cu, 15N-histidine labeled enzyme and interpretation of hyperfine couplings by molecular orbital calculations

 CW ENDOR (X-band) spectra for the purple mixed-valence [Cu(1.5+)...Cu(1.5+)], S = 1/2, Cu_A site in nitrous oxide reductase were obtained after insertion of ⁶⁵Cu or both ⁶⁵Cu and ¹⁵N-histidine. The ¹⁴N/¹⁵N isotopic substitution allowed for an unambiguous deconvolution of proton and nitrogen hyperfine couplings in the spectra. A single nitrogen coupling with a value of 12.9 ± 0.4 MHz for ¹⁴N was detected. Its anisotropy was characteristic for imidazole bound to copper. A spin density of 3–5% was estimated for the nitrogen donors to Cu_A, indicating that the ground state is ²B_3u. Proton hyperfine structure was detected from four C_β protons of coordinating cysteine residues. Their isotropic and anisotropic parts were deconvoluted by spectral simulation. From the anisotropic couplings a spin density of 16–24% was estimated for each of the cysteine thiolate donors of Cu_A. The [N_HisCu(RS)₂CuN_His]⁺ core structure of Cu_A in nitrous oxide reductase from Pseudomonas stutzeri is predicted to be similar to the crystallographically determined Cu_A* structure (Wilmanns M, Lappalainen P, Kelly M, Sauer-Eriksson E, Saraste M (1995) Proc Natl Acad Sci USA 92 : 11955–11959), but distinct from the Cu_A structure of Paracoccus denitrificans cytochrome c oxidase (Iwata S, Ostermeier C, Ludwig B, Michel H (1995) Nature 376 : 660–669). The angular dependence of the isotropic couplings as a function of the electronic ground state was calculated by the INDO/S method. The Mulliken atomic-spin populations calculated by a gradient-corrected density functional method and the semiempirical INDO/S method were compared with experimentally derived spin populations, and good agreement between theory and experiment was found for both calculations. The ground state of Cu_A is best represented by the resonance structures of the form [Cu^IS^–S^–Cu^II↔ Cu^IS^•S^–Cu^I↔ Cu^IS^–S^•Cu^I↔ Cu^IIS^–S^–Cu^I]. It is proposed that the Cu 4s,p as well as sulfur 3d orbitals play a role in the stabilization of this novel type of cluster. Received: 17 September 1997 / Accepted: 28 October 1997     

N- and O-linked oligosaccharides completely lack galactose residues in the gms1och1 mutant of Schizosaccharomyces pombe

Unlike their counterparts in budding yeast Saccharomyces cerevisiae, the glycoproteins of Schizosaccharomyces pombe contain, in addition to α-d-mannose (Man), a large number of α-d-galactose (Gal) residues. In both yeasts, large outer chains are attached to the oligosaccharide cores of glycoproteins during export via Golgi. Formation of the yeast-specific large outer chain is initiated by α-1,6-mannosylatransferase encoded by the och1 ⁺ gene, the disruption of which blocked outer chain elongation. We previously reported that N-linked oligosaccharide structures of S. pombe och1Δ mutant consisted of Gal_2–6Man₉GlcNAc₂ with α-linked Gal residues attached to the core oligosaccharide moiety. The disruption of gms1 ⁺, a gene encoding the UDP-galactose transporter required for the synthesis of galactomannan, abolished cell surface galactosylation in S. pombe. In this study, we constructed a gms1Δoch1Δ double mutant and determined the N- and O-linked oligosaccharide structures present on the cell surface. Oligosaccharides were liberated from glycoproteins by hydrazinolysis and labeled with the fluorophore, 2-aminopyridine. The pyridylaminated N-linked oligosaccharides were analyzed by high-performance liquid chromatography in combination with α1,2-mannosidase digestion and partial acetolysis. These analyses revealed that the N-linked oligosaccharides of gms1Δoch1Δ cells consisted of α1,2-linked Man-extended core oligosaccharides (Man_8–12GlcNAc₂) from which the fission yeast-specific α-linked Gal residues were completely absent.     

Azide adducts of stearoyl-ACP desaturase: a model for μ-1,2 bridging by dioxygen in the binuclear iron active site

 The stearoyl-acyl carrier protein Δ⁹ desaturase (Δ9D) uses an oxo-bridged diiron center to catalyze the NAD(P)H– and O₂–dependent desaturation of stearoyl-ACP. Δ9D, ribonucleotide reductase, and methane monooxygenase have substantial similarities in their amino acid primary sequences and the physical properties of their diiron centers. These three enzymes also appear to share common features of their reaction cycles, including the binding of O₂ to the diferrous state and the subsequent generation of transient diferric-peroxo and diferryl species. In order to investigate the coordination environment of the proposed diferric-peroxo intermediate, we have studied the binding of azide to the diiron center of Δ9D using optical, resonance Raman (RR), and transient kinetic spectroscopic methods. The addition of azide results in the appearance of new absorption bands at 325 nm and 440 nm (k _app≈3.5 s^–1 in 0.7 M NaN₃, pH 7.8). RR experiments demonstrate the existence of two different adducts: an η¹–terminal structure at pH 7.8 (¹⁴N₃ ^– asymmetric stretch at 2073 cm^–1, resolved into two bands with ¹⁵N¹⁴N₂ ^–) and a μ-1,3 bridging structure at pH<7 (¹⁴N₃ ^– asymmetric stretch at 2100 cm^–1, shifted as a single band with ¹⁵N¹⁴N₂ ^–). Both adducts also exhibit an Fe–N₃ stretching mode at ≈380 cm^–1, but no accompanying Fe–O–Fe stretching mode, presumably due to either protonation or loss of the oxo bridge. The ability to form a μ-1,3 bridging azide supports the likelihood of a μ-1,2 bridging peroxide as a catalytic intermediate in the Δ9D reaction cycle and underscores the adaptability of binuclear sites to different bridging geometries. Received: 23 August 1996 / Accepted: 4 October 1996     

Sediminihabitans luteus gen. nov., sp. nov., a new member of the family Cellulomonadaceae isolated from sea sediment

Two novel Gram-positive actinobacteria, designated H97-3^T and H83-5, were isolated from marine sediment samples and their taxonomic positions were investigated by a polyphasic approach. Both strains formed vegetative hyphae in the early phase of growth but the hyphae eventually fragmented into coccoid cells. The peptidoglycan type was found to be A4α. The predominant menaquinone was MK-9(H₄), and the major fatty acids were anteiso-C_15:0, anteiso-C_17:0 and C_16:0. The DNA G+C content was 74.0–74.9 mol %. 16S rRNA gene sequencing analysis revealed that strains H97-3^T and H83-5 represented novel members of the family Cellulomonadaceae. Their nearest phylogenetic neighbours were the members of the genus Oerskovia, with a similarity of 98.3–98.4 %. However, strains H97-3^T and H83-5 were distinguishable from the members of the genus Oerskovia and the other genera of the family Cellulomonadaceae in terms of chemotaxonomic characteristics and phylogenetic relationship. The result of the DNA–DNA hybridization indicated that strains H97-3^T and H83-5 belonged to the same species. Therefore, strains H97-3^T and H83-5 represent a novel genus and species of the family Cellulomonadaceae, for which the name Sediminihabitans luteus gen. nov., sp. nov. is proposed. The type strain of S. lutes is H97-3^T (=NBRC 108568^T = DSM 25478^T).     

Structure of cellulose II hydrate

A hydrate of cellulose II can be formed by swelling Fortisan fibers in hydrazine and then washing in water. The hydrate is stable at 93% relative humidity and has a monoclinic unit cell with dimensions a = 9.02 Å, b = 9.63 Å, c = 10.34 Å, and γ = 116.0°; the space group is P2₁. The unit cell contains disaccharide sections of two chains and approximately four water molecules. The structure was refined using the LALS method, based on 10 observed and 10 unobserved reflections. An antiparallel arrangement of adjacent chains was assumed, since this occurs in cellulose II (the starting material), and the hydrate also reverts to cellulose II on dehydration. Refinement of the positions and side-chain conformations of the chains shows that the chains are stacked in the same way as in cellulose II, and the hydrate is formed by insertion of water molecules between the stacks. However, all efforts to arrange the water molecules in crystallographically regular positions led to unsatisfactory agreement between the observed and calculated intensities. These results suggest an irregular arrangement of the water molecules, which was modeled using water-weighted atomic scattering factors. The analysis resulted in two refined models with relative chain staggers of ～ +c/4 and ～ -c/4, which are indistinguishable in terms of the x-ray agreement. Our preference is for the +c/4 model, for which the stacks of chains are analogous to those in cellulose II.