Quantitative NMR using water as internal calibrant.

A new quantitative nuclear magnetic resonance (qNMR) method, called qNMRw, using water as the internal calibrant has been developed. Its principles, procedures, calculations, and test results are presented here. It is shown to avoid the difficulties created by moisture present in other reference materials. High precision and accuracy can be achieved with qNMRw. The method can be used for analyzing technical materials, herbicide formulation products, and other types of chemical samples. It can also be used to measure the purity and concentration of materials to be used as quantitation calibrants.

Structural and functional characterization of triketone dioxygenase from Oryza Sativa.

The transgenic expression of rice triketone dioxygenase (TDO; also known as HIS1) can provide protection from triketone herbicides to susceptible dicot crops such as soybean. Triketones are phytotoxic inhibitors of plant hydroxyphenylpyruvate dioxygenases (HPPD). The TDO gene codes for an iron/2-oxoglutarate-dependent oxidoreductase. We obtained an X-ray crystal structure of TDO using SeMet-SAD phasing to 3.16 Å resolution. The structure reveals that TDO possesses a fold like that of Arabidopsis thaliana 2-oxoglutarate­iron-dependent oxygenase anthocyanidin synthase (ANS). Unlike ANS, this TDO structure lacks bound metals or cofactors, and we propose this is because the disordered flexible loop over the active site is sterically constrained from folding properly in the crystal lattice. A combination of mass spectrometry, nuclear magnetic resonance, and enzyme activity studies indicate that rice TDO oxidizes mesotrione in a series of steps; first producing 5-hydroxy-mesotrione and then oxy-mesotrione. Evidence suggests that 5-hydroxy-mesotrione is a much weaker inhibitor of HPPD than mesotrione, and oxy-mesotrione has virtually no inhibitory activity. Of the close homologues which have been tested, only corn and rice TDO have enzymatic activity and the ability to protect plants from mesotrione. Correlating sequence and structure has identified four amino acids necessary for TDO activity. Introducing these four amino acids imparts activity to a mesotrione-inactive TDO-like protein from sorghum, which may expand triketone herbicide resistance in new crop species.

In-gel protein N- and C-termini identification and its application for transgenic protein characterization.

A new method for the determination of N- and C-termini of a protein isolated in a polyacrylamide gel is introduced. In-gel partial protein hydrolysis by hydrochloric acid is used to generate N- and C-terminal peptides for identification. This new method is complementary to existing techniques. The application of the in-gel protein termini identification technique to the characterization of the transgenic protein diacylglycerol acyltransferase (UrDGAT2A) purified from soybean seeds is also reported here. Both N- and C-termini of UrDGAT2A were successfully identified and the N-terminus was found to be blocked by acetylation. The analysis results of UrDGAT2A and two commercial proteins (bovine serum albumin (BSA) and alcohol dehydrogenase) are used to demonstrate the effectiveness of the method in identifying actual N- and C-termini, terminal truncation and blocking.

The entomopathogenic bacterial endosymbionts Xenorhabdus and Photorhabdus: convergent lifestyles from divergent genomes.

Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points.

An accurate and clean calibration method for MALDI-MS.

A new method for the mass calibration of the matrix-assisted laser desorption/ionization-mass spectrometry spectrum is introduced. This method achieves the same accuracy as that of internal calibration but without its drawbacks. The interference and signal suppression by calibration standard are avoided, and a pure/clean sample spectrum is obtained. No prior knowledge about the sample quantity is required for the calibration. The effectiveness of the method is demonstrated with protein identification data.

The Dean-Evans relation in (31)P NMR spectroscopy and its application to the chemistry of octahedral tungsten sulfide clusters.

One of the challenges in studying the chemistry of hexanuclear octahedral metal clusters is analyzing the many possible complexes, including stereoisomers, when these complexes consist of mixed axial ligands (two or more). In the case of W(6)S(8)L(6-n)(PR(3))(n)(n = 0-6; L = nonphosphine Lewis base ligands, PR(3) = phosphines) clusters, in situ identification of the 10 possible complexes is possible by (31)P NMR due to P-W-W-P coupling. A linear relation for (31)P NMR shifts (delta((31)P)) of these W(6)S(8)L(6-n)(PR(3))(n) complexes, analogous to the Dean-Evans relation for (19)F NMR shifts of octahedral tin complexes, is found and expressed as delta((31)P) = delta(ref) + pC + qT with two variables (p and q, the number of ligands L in the cis or trans position to PR(3), respectively) with two constants (C and T, characteristic of a given ligand L). (31)P NMR investigation of over 200 complexes in 26 W(6)S(8)L(6-n)(PR(3))(n) systems show that this relation is generally valid for W(6)S(8) clusters. Such a relation helps spectroscopic assignments and demonstrates the trans and cis influence on hexanuclear clusters. Large bulky ligands cause deviations from the linear behavior due to steric effects. With the help of 2-D (31)P NMR spectroscopy, mixtures of W(6)S(8)(PR(3))(6-n)(PR'(3))(n) (n = 0-6) complexes can also be unequivocally interpreted. The Dean-Evans relation is expanded to account for different phosphine ligands. Partial substitution reactions of these W(6)S(8) complexes by phosphines were investigated using (31)P NMR, and four single crystals of mixed-ligand clusters are characterized with X-ray diffraction. In summary, (31)P NMR and other NMR techniques, combined with Dean-Evans relations, are invaluable analytical tools for studying molecular W(6)S(8) cluster chemistry and are likely to be useful for studying other mixed-ligand metal clusters.

Cloning, expression and characterization of a family-74 xyloglucanase from Thermobifida fusca.

Thermobifida fusca xyloglucan-specific endo-beta-1,4-glucanase (Xeg)74 and the Xeg74 catalytic domain (CD) were cloned, expressed in Escherichia coli, purified and characterized. This enzyme has a glycohydrolase family-74 CD that is a specific xyloglucanase followed by a family-2 carbohydrate binding module at the C terminus. The Michaelis constant (Km) and maximal rate (Vmax) values for hydrolysis of tamarind seed xyloglucan (tamXG) are 2.4 micro m and 966 micro mol xyloglucan oligosaccharides (XGOs) min-1. micro mol protein-1. More than 75% of the activity was retained after a 16-h incubation at temperatures up to 60 degrees C. The enzyme was most active at pH 6.0-9.4. NMR analysis showed that its catalytic mechanism is inverting. The oligosaccharide products from hydrolysis of tamXG were determined by MS analysis. Cel9B, an active carboxymethylcellulose (CMC)ase from T. fusca, was also found to have activity on xyloglucan (XG) at 49 micro mol.min-1. micro mol protein-1, but it could not hydrolyze XG units containing galactose. An XG/cellulose composite was prepared by growing Gluconacetobacterxylinus on glucose with tamXG in the medium. Although a mixture of purified cellulases was unable to degrade this material, the composite material was fully hydrolyzed when Xeg74 was added. T. fusca was not able to grow on tamXG, but Xeg74 was found in the culture supernatant at the same level as was found in cultures grown on Solka Floc. The function of this enzyme appears to be to break down the XG surrounding cellulose fibrils found in biomass so that T. fusca can utilize the cellulose as a carbon source.

Distortion of the three-dimensional structure of the vnd/NK-2 homeodomain bound to DNA induced by an embryonically lethal A35T point mutation.

The three-dimensional solution structure obtained by NMR of the A35T mutant vnd/NK-2 homeodomain bound to the vnd/NK-2 consensus 16 bp DNA sequence was determined. This mutation to threonine from alanine in position 35 in helix II of the vnd/NK-2 homeodomain is associated with early embryonic lethality in Drosophila melanogaster. Although the unbound mutant protein is not structured, in the DNA-bound state it adopts the three-helix fold characteristic of all known homeodomains, but with alterations relative to the structure of the wild-type analogue. These structural modifications occur, and are accompanied by a 50-fold reduction in the DNA binding affinity, even though most of the protein-DNA interactions originally seen for the wild-type homeodomain are found likewise in the threonine analogue. Alterations include torsional angle changes in the loop between helix I and helix II, and in the turn between helix II and helix III, as well as in a distortion of the usual antiparallel orientation of helix I with respect to helix II. The alteration of the position of leucine 40 in the A35T mutant is proposed to explain the observed 1.27 ppm upfield shift of the corresponding amide proton resonance relative to the value observed for the wild-type analogue. A detailed comparison of the structures of the mutant A35T and wild-type vnd/NK-2 homeodomains bound to the cognate DNA is presented. The consequences of the structural alteration of the DNA-bound A35T mutant vnd/NK-2 protein may constitute the basis of the observed early embryonic lethality.

New non-glycosidic diterpenes from the leaves of Stevia rebaudiana.

Six new labdane-type, non-glycosidic diterpenes, sterebins I-N (1-6), were isolated from the leaves of Stevia rebaudiana. Their structures, analogous to those of the previously described sterebins A-H, were elucidated on the basis of spectroscopic and chemical studies.