Klebsiella phage KP34gp57 capsular depolymerase structure and function: from a serendipitous finding to the design of active mini-enzymes against K. pneumoniae.

IMPORTANCE: In this work, we determined the structure of Klebsiella phage KP34p57 capsular depolymerase and dissected the role of individual domains in trimerization and functional activity. The crystal structure serendipitously revealed that the enzyme can exist in a monomeric state once deprived of its C-terminal domain. Based on the crystal structure and site-directed mutagenesis, we localized the key catalytic residues in an intra-subunit deep groove. Consistently, we show that C-terminally trimmed KP34p57 variants are monomeric, stable, and fully active. The elaboration of monomeric, fully active phage depolymerases is innovative in the field, as no previous example exists. Indeed, mini phage depolymerases can be combined in chimeric enzymes to extend their activity ranges, allowing their use against multiple serotypes.

Studies on the efficient generation of phosphorus-carbon bonds via a rearrangement of P(III) esters catalysed by trimethylhalosilanes.

Halotrimethylsilanes Me(3)SiX (X = Br, I) catalyse rearrangements of tricoordinate phosphorus esters R'R''P-OR into the corresponding phosphoryl systems R'R''P(O)R. This provides a simple and efficient route to a variety of structures containing phosphorus-carbon bonds, under mild conditions and with good yields. The reaction mechanism was investigated in detail by (31)P NMR spectroscopy and independent synthesis of the reaction intermediates. It has been demonstrated that the primary products of this catalytic reaction are halogeno P(III) structures R'R''PX and silyl ethers ROSiMe(3) and that they subsequently react to give the corresponding phosphorus silyl esters-Me(3)SiOPR'R''-and alkyl halides RX. At higher temperatures these intermediates then react to form R'R''P(O)R compounds. This paper also features the surprising observation that when esters Ph(2)POR and halotrimethylsilanes Me(3)SiX (X = Br, I) are used in 2:1 ratio, phosphonium salts Ph(2)R(2)P(+)X(-) and trimethylsilyl diphenylphosphinate--Ph(2)P(O)OSiMe(3)--are formed as the major products. Experimental evidence indicates that the mechanisms of both reactions are fundamentally different from that of the Michaelis-Arbuzov reaction. Me(3)SiCl is not reactive and this paper explains why.

Solid-state NMR spectroscopy as a tool supporting optimization of MALDI-TOF MS analysis of polylactides.

We report systematic structural studies of poly(l-lactide) (PLLA) employing matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and solid-state NMR spectroscopy. (13)C cross polarization magic angle spinning (CP/MAS) NMR data for 1,8-dihydroxy-9-anthracenone (DT), 2,5-dihydroxybenzoic acid (DHB), 2-(4-hydroxyphenylazo)-benzoic acid (HABA), and trans-3-indoleacrylic acid (IAA), four matrices commonly used in MALDI-TOF analysis of polymers, were analyzed to test the influence of crystallization conditions (solvent, inorganic salt) on sample morphology. (13)C principal elements of chemical shift tensor (CST) and line-shape analyses were employed to study of the nature of hydrogen bonding and to evaluate the crystallinity and amorphicity of the pure polymer. NMR parameters for PLLA were compared with data for polylactide crystallized with the four matrices under different conditions with the addition of two inorganic salts as cationization agents. This study revealed that the semicrystalline structure of the polymer does not change when it is embedded in the matrix.

(15)N and (13)C high-resolution solid-state NMR study of the polymorphism of the L-enantiomer of N-benzoylphenylalanine.

In this paper, several approaches which allow the investigation of mixtures of polymorphs, employing modern solid-state NMR (SS NMR) spectroscopy are reported. A convenient methodology for characterization of the hydrogen bonding and molecular conformation of a polymorphic sample by means of one-dimensional and two-dimensional, 13C and 15N NMR experiments as well as CSA tensor analysis and theoretical calculations is presented. Two-dimensional heteronuclear SS NMR allowed definition of the polymorphic domain of N-benzoyl-L-phenylalanine (N-Bz-Phe). The graphical method of Herzfeld and Berger was used to measure the 13C and 15N spinning sideband intensities which allowed the calculation of NMR parameters for labeled centers of N-Bz-Phe. The experimental data were compared with computed results obtained by means of the DFT hybrid method with B3PW91 functional and 6-311++G** basis set.

31P double-quantum solid-state NMR study of phosphoroorganic compounds with (O)P-O-P-(O), (S)P-O-P(S) and (S)P-S-P(O) unit.

In this work we have tested applicability of the commonly used double quantum recoupling sequence POST-C7 to study of (31)P-(31)P geometrical constraints for phosphoroorganic model compounds with different chemical shift anisotropy (CSA) and distinct molecular dynamics in the crystal lattice. Our results clearly show that even with large CSA, POST-C7 gives good efficiency of (31)P double-quantum excitations. Moreover, large amplitude molecular motion only slightly disturb (31)P build-up curve. Chi(2) error analysis is used for verification of values and orientations of chemical shift tensors (CST) parameters employed for simulation of POST-C7 buildup curves.

The determination of enantiomeric excess of valine by ODESSA solid-state NMR experiment.

The enantiomeric excess (ee) can be determined by many methods; one among them is nuclear magnetic resonance in solid-state (SS NMR). In this study we used the SS NMR ODESSA experiment for determination of the ee of valine.

Study of molecular dynamics and the solid state phase transition mechanism for unsymmetrical thiopyrophosphate using X-ray diffraction, DFT calculations and NMR spectroscopy.

Differential scanning calorimetry (DSC) and low-temperature X-ray diffraction studies showed that 2-thio-(5,5-dimethyl-1,3,2-dioxaphosphorinanyl)2'-oxo-dineopentyl-thiophosphate (compound 1) undergoes reversible phase transition at 203 K related to the change of symmetry of the crystallographic unit. Solid state NMR spectroscopy was used to establish the dynamic processes of aliphatic groups and the phosphorus skeleton. 13C and 31P variable temperature NMR studies as well as T1 and T1rho measurements of relaxation times revealed the different mode of molecular motion for each neopentyl residue directly bonded to phosphorus. It is concluded that molecular dynamics of aliphatic groups causes different van der Waals interactions in the crystal lattice and is the driving force of phase transition for compound 1. Finally, we showed that very sharp phase transition temperature makes compound 1 an excellent candidate as a low-temperature NMR thermometer in the solid phase.

Probing structure in the polymorphic domain of the L-enantiomer of N-benzoyl-phenylalanine by means of 2D solid-state NMR spectroscopy and DFT calculations.

A study of polymorphism using a range of solid-state NMR techniques is presented. We demonstrate the existence of at least six polymorphs in a sample of N-benzoyl-L-phenylalanine. We also present methodology for the characterization of the protonation state, hydrogen bonding, and molecular conformation for the polymorphs, together with results of such a characterization for one of the polymorphs present in our sample. DFT modeling is used to investigate the separate effects hydrogen bonding and molecular conformation have on the chemical shift tensor.

Solid state NMR studies and density functional theory (DFT) calculations of conformers of quercetin.

This work presents the first comparative analysis of two crystallographic modifications of quercetin (3,3',4',5,7-penta-hydroxyflavone). The existence of dihydrate and unhydrated forms of quercetin in the solid state is confirmed by several experimental techniques e.g. X-ray diffraction of powders, DSC, TGA, and NMR. Our studies allow an understanding of the complexity of quercetin samples obtained from different sources. A PASS-2D experiment is employed to establish principal values of 13C chemical shift tensors for both modifications. Solid state NMR spectroscopy and DFT GIAO calculations provide unique information about NMR shielding and electron density distribution for different conformers. It has been concluded that changes of conformation and hydrogen bonding pattern have great influence on bond order parameters of quercetin. Theoretical calculations and experimental data do not exclude the existence of the syn conformer of quercetin, which so far was not considered in the condensed phase.

An experimental and theoretical study of the 13C and 31P chemical shielding tensors in solid O-phosphorylated amino acids.

A series of l and dl forms of O-phosphorylated amino acids (serine, threonine, tyrosine) have been studied by using solid-state multinuclear NMR spectroscopy and ab initio calculations. Principal elements of the (13)C and (31)P chemical shielding tensors have been measured and discussed in relation to zwitterionic structures and intermolecular contacts. DFT calculations have been compared with experimental data showing their ability to reproduce experimentally obtained tensor values in this challenging class of compounds. The changes of orientation of (31)P chemical shielding tensor with respect to the molecular frame in the presence of hydrogen bonds have been revealed and discussed on the ground of theoretical calculations. The measurements of internuclear P...P distances, based on Zeeman magnetization exchange between (31)P spins with differing chemical shielding tensor orientations, were exploited for a clear distinction between enantiomers and racemates.

X-ray, 31P CP/MAS, and single-crystal NMR studies, and 31P DFT GIAO calculations of inclusion complexes of bis[6-O,6-O'-(1,2:3,4- diisopropylidene-alpha-D-galactopyranosyl)thiophosphoryl] disulfide: the importance of C-H...S=P contacts in the solid state.

Bis[6-O,6-O'-(1,2:3,4-diisopropylidene-alpha-D-galactopyranosyl) thiophosphoryl] disulfide shows a strong tendency to form inclusion compounds. The crystal and molecular structure of eight different solvates was established by X-ray analysis. The results indicate three different types of disulfide arrangements in the crystal lattice. By means of 31P CP/MAS NMR experiments the principal values delta 11, delta 22, and delta 33 of the 31P chemical shift tensor were obtained for each form. The orientation of its principal axes with respect to a molecular frame was investigated by means of 31P CP and single-crystal NMR for the complex with propan-2-ol. The principal axis 1 of both chemically equivalent phosphorus atoms is nearly parallel to the P-S bond and the principal axis 3 is very close to the P=S bond. DFT GIAO calculations of the model compound (EtO)2(S)P1SSP2(S)-(OEt)2 allowed assignment of the experimental chemical shift curves to the magnetically nonequivalent atoms P1 and P2. The maximum difference between calculated angles [symbol: see text] i-P-X)calcd and experimental angles [symbol: see text] i-P-X)exptl is 8.3 degrees and the rms distance 3.8 degrees (i = principal axes 1, 2, 3; X = S, -S-, -O1-, -O2-). The influence of C-H...S weak hydrogen bonding on phosphorus shielding was tested theoretically (31P DFT GIAO) employing the dimethoxythiophosphoryl disulfide.CH4 complex as a model compound. The sensitivity of 31P delta ii parameters to intermolecular forces is demonstrated.