Jeremy N. S. Evans

The solution structure of an HMG-I(Y)-DNA complex defines a new architectural minor groove binding motif.

The solution structure of a complex between a truncated form of HMG-I(Y), consisting of the second and third DNA binding domains (residues 51–90), and a DNA dodecamer containing the PRDII site of the interferon-β promoter has been solved by multidimensional nuclear magnetic resonance spectroscopy. The stoichiometry of the complex is one molecule of HMG-I(Y) to two molecules of DNA. The structure reveals a new architectural minor groove binding motif which stabilizes B-DNA, thereby facilitating the binding of other transcription factors in the opposing major groove. The interactions involve a central Arg-Gly-Arg motif together with two other modules that participate in extensive hydrophobic and polar contacts. The absence of one of these modules in the third DNA binding domain accounts for its ∼100 fold reduced affinity relative to the second one.

Purification, Cloning, and Heterologous Expression of a Catalytically Efficient Flavonol 3-O-Galactosyltransferase Expressed in the Male Gametophyte of Petunia hybrida

Flavonols are plant-specific molecules that are required for pollen germination in maize and petunia. They existin planta as both the aglycone and glycosyl conjugates. We identified a flavonol 3-O-galactosyltransferase (F3GalTase) that is expressed exclusively in the male gametophyte and controls the formation of a pollen-specific class of glycosylated flavonols. Thus an essential step to understanding flavonol-induced germination is the characterization of F3GalTase. Amino acid sequences of three peptide fragments of F3GalTase purified from petunia pollen were used to isolate a full-length cDNA clone. RNA gel blot analysis and enzyme assays confirmed that F3GalTase expression is restricted to pollen. Heterologous expression of the F3GalTase cDNA inEscherichia coli yielded active recombinant enzyme (rF3GalTase) which had the identical substrate specificity as the native enzyme. Unlike the relatively nonspecific substrate usage of flavonoid glycosyltransferases from sporophytic tissues, F3GalTase uses only UDP-galactose and flavonols to catalyze the formation of flavonol 3-O-galactosides. Kinetic analysis showed that thek cat/K m values of rF3GalTase, using kaempferol and quercetin as substrates, approaches that of a catalytically perfect enzyme. rF3GalTase catalyzes the reverse reaction, generation of flavonols from UDP and flavonol 3-O-galactosides, almost as efficiently as the forward reaction. The biochemical characteristics of F3GalTase are discussed in the context of a role in flavonol-induced pollen germination.

Structural studies of Streptococcus pneumoniae EPSP synthase in unliganded state, tetrahedral intermediate-bound state and S3P-GLP-bound state.

The shikimate pathway synthesizes aromatic amino acids and other essential metabolites that are necessary for bacteria, plants and fungi to survive. This pathway is not present in vertebrates and therefore represents an attractive target for antibacterial agents. We have successfully crystallized and solved the structure of unliganded, inhibitor‐liganded and tetrahedral intermediate (TI)‐liganded forms of Streptococcus pneumoniae EPSP synthase. The overall topology of the S. pneumoniae EPSP synthase is similar to that of the Escherichia coli EPSP synthase. In addition, the majority of residues responsible for ligand binding were conserved between the two proteins. TI‐liganded structure provides absolute configuration of the C‐2 atom from the F‐PEP moiety of the enzyme‐bound intermediate and also defines key residues responsible for the enzyme reaction. Comparison of the unliganded state and substrate‐bound state of the enzyme provides insights into the structural mechanisms involved in dynamic events of ligand binding, domain movement and closure. This structural study of the pathogenic bacteria S. pneumoniae EPSP synthase with inhibitor and TI will provide invaluable information for the design of new‐generation antibiotics.

Placement of 19F into the center of GB1: effects on structure and stability

A structural and thermodynamic characterization of 5F‐Trp‐substituted immunoglobulin binding domain B1 of streptococcal protein G (GB1) was carried out by nuclear magnetic resonance and circular dichroism spectroscopy. A single fluorine reporter atom was positioned at the center of the three‐dimensional structure, uniquely poised to be exploited for studying interior properties of this protein. We demonstrate that the introduction of 5F‐Trp does not affect the global and local architecture of GB1 and has no influence on the thermodynamic stability. The favorable properties of the fluorinated GB1 render this molecule a desirable model system for the development of spectroscopic methodology and theoretical calculations.

Effects of sample preparation conditions on biomolecular solid-state NMR lineshapes

Sample preparation conditions with the 46 kDa enzyme complex of 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, shikimate-3-phosphate (S3P) and glyphosate (GLP) have been examined in an attempt to reduce linewidths in solid-state NMR spectra. The linewidths of 13P resonances associated with enzyme bound S3P and GLP in the lyophilized ternary complex have been reduced to 150 ± 12 Hz and 125 ± 7 Hz respectively, by a variety of methods involving additives and freezing techniques.

Improved heteronuclear decoupling in REDOR with the use of TPPM

Abstract Rotational-echo double-resonance (REDOR) solid-state NMR spectroscopy exhibits an explicit proton radio frequency (RF) decoupling power dependence. Experimental data using model compounds are shown that demonstrate this; as proton RF decoupling powers are increased, the REDOR difference curves change in shape with the maximum REDOR difference approaching unity. This is an important consideration when fitting data with simulations in an attempt to determine internuclear distances from dipole–dipole couplings. Often, the desired decoupling powers that achieve convergence of REDOR difference curves require proton RF powers outside the capabilities of conventional MAS solid-state probes. To alleviate this problem, the two-pulse phase-modulated (TPPM) pulse train is applied during the mixing period of the REDOR pulse sequence. The results show improved efficiency of proton decoupling when TPPM is employed, as compared to the continuous wave case at the same decoupling RF strength. Simulations of a four spin–1/2 system qualitatively agree with the experiments.

Time‐resolved solid‐state REDOR NMR studies of UDP N‐acetylglucosamine enolpyruvyl transferase

The new method of time‐resolved solid‐state rotational echo double resonance (REDOR) NMR spectroscopy introduced recently by this laboratory has been applied to the enzyme uridine N‐acetylglucosamine (UDP‐NAG) enolpyruvyl transferase (EPT), with the goal of probing the interactions between reactive species and their enzyme active site. The approach has been used in a qualitative fashion with the enzyme‐inhibitor and enzyme‐intermediate complexes of uniformly 15N‐labeled UDP‐NAG EPT, trapped under steady‐state and pre‐steady‐state conditions. A different set of intermolecular interactions between the substrates UDP‐NAG, UDP‐NAG plus 3‐Z‐fluorophosphoenolpyruvate, covalent O‐phosphothioketal, and UDP‐NAG plus phosphoenolpyruvate trapped under time‐resolved conditions (after 50 ms reaction time), and the EPT enzyme active site were observed, and this is contrasted to a similar study of the interactions in a related enzyme, 5‐enolpyruvyl‐shikimate‐3‐phosphate synthase.

Theoretical study of rotational‐echo double‐resonance and related experiments

This paper discusses the theory of the rotational‐echo double‐resonance (REDOR) and related experiments by using a different approach than that used by previous researchers. The theory has been used to discuss the REDOR, rotational‐echo double‐resonance total sidebands suppression (REDORTOSS), and interrupted REDOR experiments. In the case of REDORTOSS experiment, an analytical solution has been derived which ensures that the quantification of the dipolar coupling constant and therefore the measurement of the distance between two anisotropic heteroatoms can be made. An alternative REDOR method for situations in which anisotropy is a problem is also proposed, called the interrupted REDOR.

Chemical shift mapping of shikimate-3-phosphate binding to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase.

To facilitate evaluation of enzyme–ligand complexes in solution, we have isolated the 26‐kDa N‐terminal domain of 5‐enolpyruvylshikimate‐3‐phosphate (EPSP) synthase for analysis by NMR spectroscopy. The isolated domain is capable of binding the substrate shikimate‐3‐phosphate (S3P), and this letter reports the localization of the S3P binding site using chemical shift mapping. Based on the NMR data, we propose that Ser23, Arg27, Ser197, and Tyr200 are directly involved in S3P binding. We also describe changes in the observed nuclear Overhauser effects (NOEs) that are consistent with a partial conformational change in the N‐terminal domain upon S3P binding.

Letter to the Editor: Sequential assignments of the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the formation of EPSP from shikimate-3phosphate (S3P) and phosphoenolpyruvate (PEP) in the sixth step of the shikimate pathway. This pathway is absent from mammalian systems but present in plants, bacteria, fungi and parasites, making EPSP synthase a promising model for the development of new antibiotics. One suggested approach toward inhibitor design is the prevention of domain closure, an event that is necessary for catalysis. However, what triggers domain closure is in question due to conflicting crystal structures of the enzyme • S3P complex. One shows the enzyme in the open state, with S3P bound only to the N-terminal domain, while the other has S3P in contact with residues of both domains and the enzyme in the closed state. We have prepared the isolated domains of EPSP synthase in order to analyze the individual contributions of the domains to the events of substrate binding and catalysis. In particular, isolation of the N-terminal domain will allow us to differentiate between the chemical shift effects of S3P binding and those of domain closure, which will provide a very specific basis for designing inhibitors of domain closure. Here we report the backbone and side chain chemical shift assignments for the isolated N-terminal domain. The assignments have provided a basis for interpretation of H2O exchange experiments (Stauffer et al., 2001a) and chemical shift mapping of the S3P binding site (Stauffer et al., 2001b).