Biosynthetic 4,6-dehydratase gene deletion: isolation of a glucosylated jadomycin natural product provides insight into the substrate specificity of glycosyltransferase JadS.

Deletion of the biosynthetic 4,6-dehydratase gene, jadT, present in the angucycline jadomycin dideoxysugar biosynthetic pathway, led to the isolation of a novel C12 glucosylated jadomycin. JadS was identified as the catalyst responsible for glucosylation due to a loss of production of the glucosylated natural product in a ΔjadSΔjadT deletion strain. This study demonstrates that a 2,6-dideoxy-l-sugar glycosyltransferase is able to transfer d-glucose, exemplifying remarkable substrate tolerance.

Kinetic evaluation of glucose 1-phosphate analogues with a thymidylyltransferase using a continuous coupled enzyme assay.

Cps2L, a thymidylytransferase, is the first enzyme in Streptococcus pneumoniae L-rhamnose biosynthesis and an antibacterial target. We herein report the evaluation of six sugar phosphate analogues selected to further probe Cps2L substrate tolerance. A modified continuous spectrophotometric assay was employed for facile detection of pyrophosphate (PPi) released from nucleotidylyltransfase-catalysed condensation of sugar 1-phosphates and nucleoside triphosphates to produce sugar nucleotides. Additionally, experiments using waterLOGSY NMR spectroscopy were investigated as a complimentary method to evaluate binding affinity to Cps2L.

Directed evolution of new glycosynthases from Agrobacterium beta-glucosidase: a general screen to detect enzymes for oligosaccharide synthesis.

BACKGROUND: Oligosaccharide synthesis is becoming increasingly important to industry as diverse therapeutic roles for these molecules are discovered. The chemical synthesis of oligosaccharides on an industrial scale is often prohibitively complex and costly. An alternative, that of enzymatic synthesis, is limited by the difficulty of obtaining an appropriate enzyme. A general screen for enzymes that catalyze the synthesis of the glycosidic bond would enable the identification and engineering of new or improved enzymes. RESULTS: Glycosynthases are nucleophile mutants of retaining glycosidases that efficiently catalyze the synthesis of the glycosidic linkage by condensing an activated glycosyl fluoride donor with a suitable acceptor sugar. A novel agar plate-based coupled-enzyme screen was developed (using a two-plasmid system) and used to select an improved glycosynthase from a library of mutants. CONCLUSIONS: Plate-based coupled-enzyme screens of this type are extremely valuable for identification of functional synthetic enzymes and can be applied to the evolution of a range of glycosyl transferases.

Site-directed mutagenesis of putative active site residues of 5-enolpyruvylshikimate-3-phosphate synthase.

The site-directed mutagenesis of a number of proposed active site residues of 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase is reported. Several of these mutations resulted in complete loss of enzyme activity indicating that these residues are probably involved with catalysis, notably K22R, K411R, D384A, R27A, R100A, and D242A. Of those, K22R, R27A, and D384A did not bind either the substrate shikimate-3-phosphate (S3P) or glyphosate (GLP). The K411R and D242A mutants bind S3P only in the presence of GLP. The kinetic characterization of mutants R100K, K340R, and E418A, which retain activity, is reported. Of those, R100K and K340R do not accumulate enzyme intermediate of enzyme-bound product under equilibrium conditions. These residues, while not essential for catalysis, are most likely important for substrate binding. All of the mutants are shown to be correctly folded by NMR spectroscopy.

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 31P 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.

Highly potent bisphosphonate ligands for phosphoglycerate kinase.

We have synthesized a series of novel analogs of 1, 3-bisphospho-D-glyceric acid, 1,3-BPG,3 and evaluated their binding to phosphoglycerate kinase, PGK (EC 2.7.2.3). Nonscissile methanephosphonic acids replace the two phosphate monoesters of 1, 3-BPG and lead to several stable, tight-binding mimics of this intermediate species in glycolysis. Multiple fluorine substitution for hydrogen in the alpha-methylene groups of the phosphonic acid 1, 3-BPG analogs markedly improves their binding to PGK as determined by NMR analysis. The best ligands bind some 50-100 times more strongly than does the substrate 3-phospho-D-glyceric acid and show a requirement for pKa3 to be generally below 6.0, while the presence of a beta-carbonyl group seems to be of secondary importance.

On the mechanism of 5-enolpyruvylshikimate-3-phosphate synthase.

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP, a precursor for the aromatic amino acids. This paper examines a recent claim [Studelska, D. R., McDowell, L. M., Espe, M. P., Klug, C. A., and Schaefer, J. (1997) Biochemistry 36, 15555-15560] that the mechanism of EPSP synthase involves two covalent enzyme-intermediates, in complete contrast to a large body of literature that has already proven the involvement of a single noncovalent intermediate. The evidence in the paper of Studelska et al. is examined closely, and unequivocal proof is provided that those authors' NMR assignments to covalent structures are in error, and that in fact the species they observed were simply the product EPSP and a side-product EPSP ketal. Since those authors used rotational-echo double-resonance (REDOR) solid-state NMR to measure intermolecular and intramolecular distances in the proposed covalent intermediates, we have used REDOR to measure the same distances in enzyme-free and enzyme-bound preparations of purified EPSP, and enzyme-free preparations of purified EPSP ketal. The distance between the shikimate ring phosphorus atom and C8 in enzyme-free EPSP is 6.6 +/- 0.1 A, which lengthens to 7.4 +/- 0.1 A in the presence of the enzyme, and in enzyme-free EPSP ketal is 5.6 +/- 0.1 A. These are entirely consistent with those measured by Studelska et al., which were 7.5 +/- 0.5 A for a putative enzyme-enolpyruvyl species and 6.1 +/- 0.3 A for a putative enzyme-ketal species.

Synthesis and binding of stable bisubstrate ligands for phosphoglycerate kinase.

Stable bisubstrate ligands of phosphoglycerate kinase (PGK) have been synthesized with AMP or ADP conjugated to hydrolytically-stable, symmetrical analogues of 1,3-bisphosphoglycerate and their binding to yeast PGK evaluated. Their Kds decrease with net negative charge, with a penta-anionic analogue 7 showing highest affinity-in accordance with its approximation to the transition state for the reaction catalysed by PGK.