Dissecting Bottromycin Biosynthesis Using Comparative Untargeted Metabolomics

Bottromycin A₂ is a structurally unique ribosomally synthesized and post‐translationally modified peptide (RiPP) that possesses potent antibacterial activity towards multidrug‐resistant bacteria. The structural novelty of bottromycin stems from its unprecedented macrocyclic amidine and rare β‐methylated amino acid residues. The N‐terminus of a precursor peptide (BtmD) is converted into bottromycin A₂ by tailoring enzymes encoded in the btm gene cluster. However, little was known about key transformations in this pathway, including the unprecedented macrocyclization. To understand the pathway in detail, an untargeted metabolomic approach that harnesses mass spectral networking was used to assess the metabolomes of a series of pathway mutants. This analysis has yielded key information on the function of a variety of previously uncharacterized biosynthetic enzymes, including a YcaO domain protein and a partner protein that together catalyze the macrocyclization.     

The pregnane derivatives of the seeds from Dregea abyssinica (Hochst.) K. Schum. (Asclepiadacea). II. Structure determination

The total or partial structure determinations of some new components from Dregea abyssinica are reported. The four drebyssogenins (F, G, J and H) are esters of the drevogenins P ( 7 ) and D( 9 ) and have the following structures: Drebyssogenin F ( 5 ) = 11-O-acetyl-12-O-(α-hydroxyisovaleryl)-drevogenin P, drebyssogenin G ( 14 ) = 11-O-acetyl-12-O-isovaleryl-drevogenin D, drebyssogenin J ( 15 ) = 11, 12-di-O-acetyl-drevogenin D. Drebyssogenin K is a mixture of K1 ( 16 ) and K2 ( 17 ), the mono-O-tiglyl and mono-O-isovaleryl derivatives of drevogenin D respectively, with as yet undetermined position of the acyl residues. The drebyssosides 1, 2 and 3 are glycosides of trisaccharides. Drebyssoside 1 ( 2 ) is pachybiosyl-cymarosyl-drevogenin A, drebyssoside 2 ( 3 ) asclepobiosyl-cymarosyl-drevogenin A and drebyssoside 3 ( 6 ) pachybiosyl-cymarosyl-drebyssogenin F. Drebyssoside 4 is a mixture of at least two components and contains the same aglycones and the same sugar units as the drebyssosides 1 and 3, but is different from them. The full structure of the two sugars (drebyssobiose ( 25 ) and sugar T ( 24 )) has not been determined; it is only shown that both are disaccharides which contain 3-O-methyl-6-deoxy-allose linked to a 2-deoxyhexose.     

Evidence for helical structure in a tetramer of α2-8 sialic acid: unveiling a structural antigen

Characteristic H-bonding patterns define secondary structure in proteins and nucleic acids. We show that similar patterns apply for α2-8 sialic acid (SiA) in H(2)O and that H-bonds define its structure. A (15)N,(13)C α2-8 SiA tetramer, (SiA)(4), was used as a model system for the polymer. At 263 K, we detected intra-residue through-H-bond J couplings between (15)N and C8 for residues R-I-R-III of the tetramer, indicating H-bonds between the (15)N's and the O8's of these residues. Additional J couplings between the (15)N's and C2's of the adjacent residues confirm the putative H-bonds. NH groups showing this long-range correlation also experience slower (1)H/(2)H exchange. Additionally, detection of couplings between H7 and C2 for R-II and R-III implies that the conformations of the linkers between these residues are different than in the monomers. These structural elements are consistent with two left-handed helical models: 2 residues/turn (2(4) helix) and 4 residues/turn (1(4) helix). To discriminate between models, we resorted to (1)H,(1)H NOEs. The 2(4) helical model is in better agreement with the experimental data. We provide direct evidence of H-bonding for (SiA)(4) and show how H-bonds can be a determining factor for shaping its 3D structure.     

A theoretical case study of type I and type II beta-turns

NMR chemical shielding anisotropy tensors have been computed by employing a medium size basis set and the GIAO-DFT(B3LYP) formalism of electronic structure theory for all of the atoms of type I and type II beta-turn models. The models contain all possible combinations of the amino acid residues Gly, Ala, Val, and Ser, with all possible side-chain orientations where applicable in a dipeptide. The several hundred structures investigated contain either constrained or optimized phi, psi, and chi dihedral angles. A statistical analysis of the resulting large database was performed and multidimensional (2D and 3D) chemical-shift/chemical-shift plots were generated. The (1)H(alpha-13)C(alpha), (13)C(alpha-1)H(alpha-13)C(beta), and (13)C(alpha-1)H(alpha-13)C' 2D and 3D plots have the notable feature that the conformers clearly cluster in distinct regions. This allows straightforward identification of the backbone and side-chain conformations of the residues forming beta-turns. Chemical shift calculations on larger For-(L-Ala)(n)-NH(2) (n=4, 6, 8) models, containing a single type I or type II beta-turn, prove that the simple models employed are adequate. A limited number of chemical shift calculations performed at the highly correlated CCSD(T) level prove the adequacy of the computational method chosen. For all nuclei, statistically averaged theoretical and experimental shifts taken from the BioMagnetic Resonance Bank (BMRB) exhibit good correlation. These results confirm and extend our previous findings that chemical shift information from selected multiple-pulse NMR experiments could be employed directly to extract folding information for polypeptides and proteins.     

Semiautomatic sequence-specific assignment of proteins based on the tertiary structure--the program st2nmr

The sequence-specific assignment of resonances is still the most time-consuming procedure that is necessary as the first step in high-resolution NMR studies of proteins. In many cases a reliable three-dimensional (3D) structure of the protein is available, for example, from X-ray spectroscopy or homology modeling. Here we introduce the st2nmr program that uses the 3D structure and Nuclear Overhauser Effect spectroscopy (NOESY) peak list(s) to evaluate and optimize trial sequence-specific assignments of spin systems derived from correlation spectra to residues of the protein. A distance-dependent target function that scores trial assignments based on the presence of expected NOESY crosspeaks is optimized in a Monte Carlo fashion. The performance of the program st2nmr is tested on real NMR data of an alpha-helical (cytochrome c) and beta-sheet (lipocalin) protein using homology models and/or X-ray structures; it succeeded in completely reproducing the correct sequence-specific assignments in most cases using 2D and/or 15N/13C Nuclear Overhauser Effect (NOE) data. Additionally to amino acid residues the program can also handle ligands that are bound to the protein, such as heme, and can be used as a complementary tool to fully automated assignment procedures.     

Computer-aided modeling of pentachlorophenol 4-monooxygenase and site-directed mutagenesis of its active site

Homology modeling was used to construct a model of the three-dimensional structure of pentachlorophenol 4-monooxygenase (PcpB). A PSI-BLAST homology search was initially performed to identify the 3D structure of proteins homologous with PcpB. The feasibility of modeled structures of PcpB was evaluated by Verify3D, which calculated structural compatibility scores based on 3D-1D profiles. The predicted structure of PcpB had an acceptable 3D-1D self-compatibility score, beyond the incorrect fold score threshold. A PcpB-pentachlorophenol (PCP) complex was then constructed utilizing the modeled PcpB structure. After energy minimization of the complex, and successive minimizations of the system that consisted of the complex and the water layer surrounding the complex, the molecular dynamics of the system were simulated. The active-site residues of PcpB were identified on the basis of the modeled structure, and PcpB mutants were then designed to change the active site residues, expressed, and purified by affinity chromatography. The mutant activity was compared with that of the wild-type to investigate the validity of the modeled structure. The experimental results suggested that Phe85, Tyr216, and Arg235 were relevant to enzyme activity, and that Tyr397 and Phe87 were important for stabilization of the structure of PcpB.     

A new peptide isolated from a marine derived Streptomyces bacillaris

A new peptide, L-O-Lac-L-Val-D-O-Hiv-D-Val (1), consisting of D-valine, L-valine, L-lactic acid, and 3-D-hydroxyisovaleric acid, was isolated from the culture of the marine sediment derived Streptomyces bacillaris. The planar structure of compound 1 was assigned by 1D, 2D NMR and mass spectroscopic analyses. Following acid and base hydrolysis, the absolute configuration of the valine residues in 1 were determined by application of the advanced Marfey's method and the absolute configurations of hydroxy acids units were determined by a HPLC method based on Mosher's reagents.     

The Structure of a Novel Neutral Lipid A from the Lipopolysaccharide of Bradyrhizobium elkanii Containing Three Mannose Units in the Backbone

The chemical structure of the lipid A of the lipopolysaccharide (LPS) from Bradyrhizobium elkanii USDA 76 (a member of the group of slow‐growing rhizobia) has been established. It differed considerably from lipids A of other Gram‐negative bacteria, in that it completely lacks negatively charged groups (phosphate or uronic acid residues); the glucosamine (GlcpN) disaccharide backbone is replaced by one consisting of 2,3‐dideoxy‐2,3‐diamino‐D ‐glucopyranose (GlcpN3N) and it contains two long‐chain fatty acids, which is unusual among rhizobia. The GlcpN3N disaccharide was further substituted by three D ‐mannopyranose (D ‐Manp) residues, together forming a pentasaccharide. To establish the structural details of this molecule, 1D and 2D NMR spectroscopy, chemical composition analyses and high‐resolution mass spectrometry methods (electrospray ionisation Fourier‐transform ion cyclotron resonance mass spectrometry (ESI FT‐ICR MS) and tandem mass spectrometry (MS/MS)) were applied. By using 1D and 2D NMR spectroscopy experiments, it was confirmed that one D ‐Manp was linked to C‐1 of the reducing GlcpN3N and an α‐(1→6)‐linked D ‐Manp disaccharide was located at C‐4′ of the non‐reducing GlcpN3N (α‐linkage). Fatty acid analysis identified 12:0(3‐OH) and 14:0(3‐OH), which were amide‐linked to GlcpN3N. Other lipid A constituents were long (ω‐1)‐hydroxylated fatty acids with 26–33 carbon atoms, as well as their oxo forms (28:0(27‐oxo) and 30:0(29‐oxo)). The 28:0(27‐OH) was the most abundant acyl residue. As confirmed by high‐resolution mass spectrometry techniques, these long‐chain fatty acids created two acyloxyacyl residues with the 3‐hydroxy fatty acids. Thus, lipid A from B. elkanii comprised six acyl residues. It was also shown that one of the acyloxyacyl residues could be further acylated by 3‐hydroxybutyric acid (linked to the (ω‐1)‐hydroxy group).     

Multidimensional NMR spectroscopy for the study of histone H4-Ni(II) interaction

The N-terminal 30-amino acid tail of histone H4, a nuclear protein, was studied as a model for the interaction of this protein with Ni(ii) ions. The behaviour of the ends-blocked Ac-SGRGKGGKGLGKGGA(15)K(16)R(17)H(18)R(19)KVLRDNIQGIT-Am fragment towards Ni(ii) was analyzed with multidimensional NMR (1D, 2D TOCSY, NOESY) and UV-Vis spectroscopy. As expected, the coordination involved the imidazolic nitrogen of the His(18) residue and the three deprotonated amidic nitrogens of the His(18), Arg(17) and Lys(16) residues, respectively. A model for the structure of the complex was calculated from the inter-residual NOEs recorded in 2D NOESY spectra. The structure obtained shows that the interaction with the metal is responsible for deep changes in the conformation of the peptide, blocking the side chain of Arg(17) and Lys(16) residues above the coordination plane. These structural modifications may be physiologically relevant to the mechanism of nickel carcinogenesis.     

Design, synthesis and X-ray structure of protein-ligand complexes: important insight into selectivity of memapsin 2 (beta-secretase) inhibitors

Structure-based design, synthesis, and X-ray structure of protein-ligand complexes of memapsin 2 are described. The inhibitors are designed specifically to interact with S2- and S3-active site residues to provide selectivity over memapsin 1 and cathepsin D. Inhibitor 6 has exhibited exceedingly potent inhibitory activity against memapsin 2 and selectivity over memapsin 1 (>3800-fold) and cathepsin D (>2500-fold). A protein-ligand crystal structure revealed cooperative interactions in the S2- and S3-active sites of memapsin 2. These interactions may serve as an important guide to design selectivity over memapsin 1 and cathepsin D.     

Boron-induced hydrogen localization in the novel metal hydride LaNi(3)BH(x) (x = 2.5-3.0)

The crystal structure and hydrogenation properties of the intermetallic boride LaNi(3)B were investigated. The hydrogen-free compound has a novel structure with orthorhombic symmetry, space group Imma, a = 4.9698(8) A, b = 7.1337(8) A, c = 8.3001(9) A, and V = 294.26(7) A(3). Thermal gravimetrical analysis reveals a hydride phase that forms near ambient conditions within the compositional range LaNi(3)BH(2.5)(-)(3.0). Single-crystal X-ray diffraction on both the alloy and the hydride, using the same crystal, shows an expansion in the a-c plane (by up to approximately 8%) and a contraction along b (by approximately 3%), while the symmetry changes from Imma to Bmmb (Cmcm) and the unit cell doubles along a and b. The cell parameters for the composition of LaNi(3)BD(2.73(4)) are a = 10.7709(7) A, b = 16.0852(10) A, c = 7.6365(5) A, V = 1323.03(15) A(3), and space group Cmcm. Four nearly fully occupied interstitial hydrogen sites were located by neutron powder diffraction on deuterides and found to have tetrahedral, La(2)Ni(2) (D1,D2), trigonal-prismatic, La(3)Ni(3) (D3), and trigonal-bipyramidal, La(2)Ni(3) (D4), metal environments. The structure can also be described in terms of alternating quasi two-dimensional [NiD](-) slabs (Ni-D = 1.62-1.97 A) and La-B sheets for which bond-valence sums suggest the limiting formula La(3+)B(0)[Ni(3)D(3)](3)(-). The La-B planes do not accommodate deuterium; the B-D and D-D interactions appear to be repulsive. The shortest B-D and D-D contacts are 2.52(2) and 2.33(2) A, respectively.     

Probing the role of the C-H...O hydrogen bond stabilized polypeptide chain reversal at the C-terminus of designed peptide helices. Structural characterization of three decapeptides

The structural characterization in crystals of three designed decapeptides containing a double d-segment at the C-terminus is described. The crystal structures of the peptides Boc-Leu-Aib-Val-Xxx-Leu-Aib-Val-(D)Ala-(D)Leu-Aib-OMe, (Xxx = Gly 2, (D)Ala 3, Aib 4) have been determined and compared with those reported earlier for peptide 1 (Xxx = Ala) and the all l analogue Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-OMe, which yielded a perfect right-handed alpha-helical structure. Peptides 1 and 2 reveal a right-handed helical segment spanning residues 1 to 7, ending in a Schellman motif with (D)Ala(8) functioning as the terminating residue. Polypeptide chain reversal occurs at residue 9, a novel feature that appears to be the consequence of a C-H.O hydrogen bond between residue 4 C(alpha)H and residue 9 CO groups. The structures of peptides 3 and 4, which lack the pro R hydrogen at the C(alpha) atom of residue 4, are dramatically different. Peptide 3 adopts a right-handed helical conformation over the 1 to 7 segment. Residues 8 and 9 adopt alpha(L) conformations forming a C-terminus type I' beta-turn, corresponding to an incipient left-handed twist of the polypeptide chain. In peptide 4, helix termination occurs at Aib(6), with residues 6 to 9 forming a left-handed helix, resulting in a structure that accommodates direct fusion of two helical segments of opposite twist. Peptides 3 and 4 provide examples of chiral residues occurring in the less favored sense of helical twist; (D)Ala(4) in peptide 3 adopts an alpha(R) conformation, while (L)Val(7) in 4 adopts an alpha(L) conformation. The structural comparison of the decapeptides reported here provides evidence for the role of specific C-H.O hydrogen bonds in stabilizing chain reversals at helix termini, which may be relevant in aligning contiguous helical and strand segments in polypeptide structures.     

Hydrogen-bonded Three-Dimensional Networks Encapsulating One-dimensional Covalent Chains: [Cu(3-ampy)(H2O)4](SO4)·(H2O) (3-ampy = 3-Aminopyridine)

A three-dimensional complex [Cu(3-ampy)(H2O)4](SO4)·(H2O) (3-ampy = 3-aminopyridine) has been synthesized. Crystallographic data: C5H16CuN2O9S, Mr = 343.80, triclinic, space group P, a = 7.675(2), b = 8.225(3), c = 10.845(3) (A), α= 86.996(4), β = 76.292(4),γ = 68.890(4)°, V = 620.0(3) (A)3, Z = 2, Dc = 1.841 g/cm3, F(000) = 354 and μ = 1.971 mm-1. The structure was refined to R = 0.0269 and wR = 0.0659 for 1838 observed reflections (I ＞ 2σ(Ⅰ)). The structure consists of [Cu(3-ampy)(H2O)4]2 cations, SO42- anions and lattice water molecules. 3-Ampy acting as a bidentate bridging ligand generates a 1D covalent chain. A supramolecular 2D framework is formed through π-π stacking of pyridine rings. The lattice water molecules and SO42- anions are located between the adjacent 2D frameworks. The hydrogen bonding interactions from lattice water molecules and SO42- anions to coordinate water extend the 2D framework into a 3D network.     

Proton delivery to ferryl heme in a heme peroxidase: enzymatic use of the Grotthuss mechanism

We test the hypothesized pathway by which protons are passed from the substrate, ascorbate, to the ferryl oxygen in the heme enzyme ascorbate peroxidase (APX). The role of amino acid side chains and bound solvent is demonstrated. We investigated solvent kinetic isotope effects (SKIE) for the wild-type enzyme and several site-directed replacements of the key residues which form the proposed proton path. Kinetic constants for H(2)O(2)-dependent enzyme oxidation to Compound I, k(1), and subsequent reduction of Compound II, k(3), were determined in steady-state assays by variation of both H(2)O(2) and ascorbate concentrations. A high value of the SKIE for wild type APX ((D)k(3) = 4.9) as well as a clear nonlinear dependence on the deuterium composition of the solvent in proton inventory experiments suggest the simultaneous participation of several protons in the transition state for proton transfer. The full SKIE and the proton inventory data were modeled by applying Gross-Butler-Swain-Kresge theory to a proton path inferred from the known structure of APX. The model has been tested by constructing and determining the X-ray structures of the R38K and R38A variants and accounts for their observed SKIEs. This work confirms APX uses two arginine residues in the proton path. Thus, Arg38 and Arg172 have dual roles, both in the formation of the ferryl species and binding of ascorbate respectively and to facilitate proton transfer between the two.     

Structural prediction of a novel chitinase from the psychrophilic Glaciozyma antarctica PI12 and an analysis of its structural properties and function

The structure of psychrophilic chitinase (CHI II) from Glaciozyma antarctica PI12 has yet to be studied in detail. Due to its low sequence identity (<30?%), the structural prediction of CHI II is a challenge. A 3D model of CHI II was built by first using a threading approach to search for a suitable template and to generate an optimum target-template alignment, followed by model building using MODELLER9v7. Analysis of the catalytic insertion domain structure in CHI II revealed an increase in the number of aromatic residues and longer loops compared to mesophilic and thermophilic chitinases. A molecular dynamics simulation was used to examine the stability of the CHI II structure at 273, 288 and 300?K. Structural analysis of the substrate-binding cleft revealed a few exposed aromatic residues. Substitutions of certain amino acids in the surface and loop regions of CHI II conferred an increased flexibility to the enzyme, allowing for an adaptation to cold temperatures. A substrate binding comparison of CHI II with the mesophilic chitinase from Coccidioides immitis, 1D2K, suggested that the psychrophilic adaptation and catalytic activity at low temperatures were achieved through a reduction in the number of salt bridges, fewer hydrogen bonds and an increase in the exposure of the hydrophobic side chains to the solvent.     

Solution 1H NMR of the molecular and electronic structure of the heme cavity and substrate binding pocket of high-spin ferric horseradish peroxidase: effect of His42Ala mutation.

Solution 1H NMR has been used to assign a major portion of the heme environment and the substrate-binding pocket of resting state horseradish peroxidase, HRP, despite the high-spin iron(III) paramagnetism, and a quantitative interpretive basis of the hyperfine shifts is established. The effective assignment protocol included 2D NMR over a wide range of temperatures to locate residues shifted by paramagnetism, relaxation analysis, and use of dipolar shifts predicted from the crystal structure by an axial paramagnetic susceptibility tensor normal to the heme. The most effective use of the dipolar shifts, however, is in the form of their temperature gradients, rather than by their direct estimation as the difference of observed and diamagnetic shifts. The extensive assignments allowed the quantitative determination of the axial magnetic anisotropy, Deltachi(ax) = -2.50 x 10(-8) m(3)/mol, oriented essentially normal to the heme. The value of Deltachi(ax) together with the confirmed T(-2) dependence allow an estimate of the zero-field splitting constant D = 15.3 cm(-1), which is consistent with pentacoordination of HRP. The solution structure was generally indistinguishable from that in the crystal (Gajhede, M.; Schuller, D. J.; Henriksen, A.; Smith, A. T.; Poulos, T. L. Nature Structural Biology 1997, 4, 1032-1038) except for Phe68 of the substrate-binding pocket, which was found turned into the pocket as found in the crystal only upon substrate binding (Henriksen, A.; Schuller, D. J.; Meno, K.; Welinder, K. G.; Smith, A. T.; Gajhede, M. Biochemistry 1998, 37, 8054-8060). The reorientation of several rings in the aromatic cluster adjacent to the proximal His170 is found to be slow on the NMR time scale, confirming a dense, closely packed, and dynamically stable proximal side up to 55 degrees C. Similar assignments on the H42A-HRP mutant reveal conserved orientations for the majority of residues, and only a very small decrease in Deltachi(ax) or D, which dictates that five-coordination is retained in the mutant. The two residues adjacent to residue 42, Ile53 and Leu138, reorient slightly in the mutant H42A protein. It is concluded that effective and very informative 1H NMR studies of the effect of either substrate binding or mutation can be carried out on resting state heme peroxidases.     

A Neutron Diffraction Study for the Crystal Structure of the Deuterium（hydrogen） L－Arginine Phosphate Monohydrate

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Surface and dynamic structures of bacteriorhodopsin in a 2D crystal, a distorted or disrupted lattice, as revealed by site-directed solid-state 13C NMR

The 3D structure of bacteriorhodopsin (bR) obtained by X-ray diffraction or cryo-electron microscope studies is not always sufficient for a picture at ambient temperature where dynamic behavior is exhibited. For this reason, a site-directed solid-state 13C NMR study of fully hydrated bR from purple membrane (PM), or a distorted or disrupted lattice, is very valuable in order to gain insight into the dynamic picture. This includes the surface structure, at the physiologically important ambient temperature. Almost all of the 13C NMR signals are available from [3-13C]Ala or [1-13C]Val-labeled bR from PM, although the 13C NMR signals from the surface areas, including loops and transmembrane alpha-helices near the surface (8.7 angstroms depth), are suppressed for preparations labeled with [1-13C]Gly, Ala, Leu, Phe, Tyr, etc. due to a failure of the attempted peak-narrowing by making use of the interfered frequency of the frequency of fluctuation motions with the frequency of magic angle spinning. In particular, the C-terminal residues, 226-235, are present as the C-terminal alpha-helix which is held together with the nearby loops to form a surface complex, although the remaining C-terminal residues undergo isotropic motion even in a 2D crystalline lattice (PM) under physiological conditions. Surprisingly, the 13C NMR signals could be further suppressed even from [3-13C]Ala- or [1-13C]Val-bR, due to the acquired fluctuation motions with correlation times in the order of 10(-4) to 10(-5) s, when the 2D lattice structure is instantaneously distorted or completely disrupted, either in photo-intermediate, removed retinal or when embedded in the lipid bilayers.     

日本续断中新的双糖链三萜皂苷的结构研究

从日本续断根部的乙醇提取物中分得一个新的三萜皂苷。经过测定,它为：3-O-α-L-吡喃鼠李糖(1→3)-β-D-吡喃葡萄糖(1→3)-α-L-吡喃鼠李糖(1→2)-α-L-吡喃阿拉伯糖9常春藤苷元－28-O-β-吡喃葡萄糖酯苷)。结果表明,采用一维SEMDY,旋转坐标NOE差谱和选择性远程DEPT核磁共振新技术相结合的方法测定糖链结构不需要对化合物进行化学降解或衍生化,方法简便、快速,测定结果可靠,每个糖基的信号可以分辨和明确归属。     

Disentangling Unusual Catalytic Properties and the Role of the [4Fe-4S] Cluster of Three Endonuclease III from the Extremophile D. radiodurans

Endonuclease III (EndoIII) is a bifunctional DNA glycosylase with specificity for a broad range of oxidized DNA lesions. The genome of an extremely radiation- and desiccation-resistant bacterium, Deinococcus radiodurans, possesses three genes encoding for EndoIII-like enzymes (DrEndoIII1, DrEndoIII2 and DrEndoIII3), which reveal different types of catalytic activities. DrEndoIII2 acts as the main EndoIII in this organism, while DrEndoIII1 and 3 demonstrate unusual and no EndoIII activity, respectively. In order to understand the role of DrEndoIII1 and DrEndoIII3 in D. radiodurans, we have generated mutants which target non-conserved residues in positions considered essential for classic EndoIII activity. In parallel, we have substituted residues coordinating the iron atoms in the [4Fe-4S] cluster in DrEndoIII2, aiming at elucidating the role of the cluster in these enzymes. Our results demonstrate that the amino acid substitutions in DrEndoIII1 reduce the enzyme activity without altering the overall structure, revealing that the residues found in the wild-type enzyme are essential for its unusual activity. The attempt to generate catalytic activity of DrEndoIII3 by re-designing its catalytic pocket was unsuccessful. A mutation of the iron-coordinating cysteine 199 in DrEndoIII2 appears to compromise the structural integrity and induce the formation of a [3Fe-4S] cluster, but apparently without affecting the activity. Taken together, we provide important structural and mechanistic insights into the three EndoIIIs, which will help us disentangle the open questions related to their presence in D. radiodurans and their particularities.