Home About us Contact
|
Home About us Contact | ||
|
|
||
First Committed Step (first + committed_step)
Selected AbstractsFunctional analysis of disease-causing mutations in human galactokinaseFEBS JOURNAL, Issue 8 2003David J. Timson Galactokinase (EC 2.7.1.6) catalyzes the first committed step in the catabolism of galactose. The sugar is phosphorylated at position 1 at the expense of ATP. Lack of fully functional galactokinase is one cause of the inherited disease galactosemia, the main clinical manifestation of which is early onset cataracts. Human galactokinase (GALK1) was expressed in and purified from Escherichia coli. The recombinant enzyme was both soluble and active. Product inhibition studies showed that the most likely kinetic mechanism of the enzyme was an ordered ternary complex one in which ATP is the first substrate to bind. The lack of a solvent kinetic isotope effect suggests that proton transfer is unlikely to be involved in the rate determining step of catalysis. Ten mutations that are known to cause galactosemia were constructed and expressed in E. coli. Of these, five (P28T, V32M, G36R, T288M and A384P) were insoluble following induction and could not be studied further. Four of the remainder (H44Y, R68C, G346S and G349S) were all less active than the wild-type enzyme. One mutant (A198V) had kinetic properties that were essentially wild-type. These results are discussed both in terms of galactokinase structure-function relationships and how these functional changes may relate to the causes of galactosemia. [source] The Mycobacterium tuberculosis ino1 gene is essential for growth and virulenceMOLECULAR MICROBIOLOGY, Issue 4 2004Farahnaz Movahedzadeh Summary Inositol is utilized by Mycobacterium tuberculosis in the production of its major thiol and of essential cell wall lipoglycans. We have constructed a mutant lacking the gene encoding inositol-1-phosphate synthase (ino1), which catalyses the first committed step in inositol synthesis. This mutant is only viable in the presence of extremely high levels of inositol. Mutant bacteria cultured in inositol-free medium for four weeks showed a reduction in levels of mycothiol, but phosphatidylinositol mannoside, lipomannan and lipoarabinomannan levels were not altered. The ino1 mutant was attenuated in resting macrophages and in SCID mice. We used site-directed mutagenesis to alter four putative active site residues; all four alterations resulted in a loss of activity, and we demonstrated that a D310N mutation caused loss of the active site Zn2+ ion and a conformational change in the NAD+ cofactor. [source] Polymorphism of the pig acetyl-coenzyme A carboxylase , gene is associated with fatty acid composition in a Duroc commercial lineANIMAL GENETICS, Issue 4 2009D. Gallardo Summary Acetyl-coenzyme A carboxylase , (ACACA) catalyses the first committed step in the biosynthesis of long-chain fatty acids (FA) by converting acetyl-CoA into malonyl-CoA. In pigs, the ACACA gene maps to a chromosome 12 QTL with important effects on FA composition. In the present study, we have sequenced the coding region of the pig ACACA gene in 15 pigs, identifying 21 polymorphic sites that were either synonymous or non-coding. Ten of these SNPs segregated in a Duroc commercial population (n = 350) for which lipid metabolism and meat and carcass quality trait records were available. Significant associations were found between two linked single nucleotide polymorphisms (c.4899G>A and c.5196T>C) and percentages of carcass lean, intramuscular fat, monounsaturated, saturated (myristic, palmitic and stearic) and polyunsaturated (linoleic) FAs in the longissimus thoracis et lumborum muscle, along with serum HDL-cholesterol concentration. The most important allele substitution effects were observed for the polyunsaturated/saturated FA ratio (13,21% of the phenotypic mean) as well as for the percentages of ,-6 and polyunsaturated FAs, especially linoleic acid (7,16% of the phenotypic mean). These results suggest the existence of a causal mutation, mapping to the chromosomal region containing the pig ACACA gene, with marked effects on FA composition of meat. [source] The structure and domain organization of Escherichia coli isocitrate lyaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2001K. L. Britton Enzymes of the glyoxylate-bypass pathway are potential targets for the control of many human diseases caused by such pathogens as Mycobacteria and Leishmania. Isocitrate lyase catalyses the first committed step in this pathway and the structure of this tetrameric enzyme from Escherichia coli has been determined at 2.1,Å resolution. E. coli isocitrate lyase, like the enzyme from other prokaryotes, is located in the cytoplasm, whereas in plants, protozoa, algae and fungi this enzyme is found localized in glyoxysomes. Comparison of the structure of the prokaryotic isocitrate lyase with that from the eukaryote Aspergillus nidulans reveals a different domain structure following the deletion of approximately 100 residues from the larger eukaryotic enzyme. Despite this, the active sites of the prokaryotic and eukaryotic enzymes are very closely related, including the apparent disorder of two equivalent segments of the protein that are known to be involved in a conformational change as part of the enzyme's catalytic cycle. [source] Expression, purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Bacillus anthracis in the presence of pyruvateACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2009Jarrod E. Voss Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the lysine-biosynthesis pathway in bacteria, plants and some fungi. In this study, the expression of DHDPS from Bacillus anthracis (Ba -DHDPS) and the purification of the recombinant enzyme in the absence and presence of the substrate pyruvate are described. It is shown that DHDPS from B. anthracis purified in the presence of pyruvate yields greater amounts of recombinant enzyme with more than 20-fold greater specific activity compared with the enzyme purified in the absence of substrate. It was therefore sought to crystallize Ba -DHDPS in the presence of the substrate. Pyruvate was soaked into crystals of Ba -DHDPS prepared in 0.2,M sodium fluoride, 20%(w/v) PEG 3350 and 0.1,M bis-tris propane pH 8.0. Preliminary X-ray diffraction data of the recombinant enzyme soaked with pyruvate at a resolution of 2.15,Å are presented. The pending crystal structure of the pyruvate-bound form of Ba -DHDPS will provide insight into the function and stability of this essential bacterial enzyme. [source] | |||||||||||||