Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.6.1.1 (aspartate aminotransferase)
 21,665 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We present here a radiochemical enzymatic endpoint assay for the guanine nucleotides GTP and GDP that is suitable for use with cell extracts. The major coupling enzyme used is phosphoenolpyruvate carboxykinase purified from chicken liver. The ancillary coupling enzyme, aspartate aminotransferase, was used to generate a low steady-state concentration of oxalacetate. GTP was determined by the overall conversion of [U-14C]aspartate into [14C]phosphoenolpyruvate. This reaction was also scaled-up as a preparative method for [U-14C]phosphoenolpyruvate. This was used with the same coupling enzymes in reverse to measure GDP by the formation of [14C]aspartate. The assay method was applied to isolated rat hepatocytes. The total GTP and GDP concentrations found were within the range reported by others for rat liver. The advantages of this assay are its sensitivity, specificity, and applicability to large numbers of samples.
 ...
PMID:A radiochemical enzymatic endpoint assay for GTP and GDP. 204 20

Perosamine or 4-amino-4,6-dideoxy- d-mannose is an unusual sugar found in the O-antigens of some Gram-negative bacteria such as Vibrio cholerae O1 (the causative agent of cholera) or Escherichia coli O157:H7 (the leading cause of food-borne illnesses). It and similar deoxysugars are added to the O-antigens of bacteria via the action of glycosyltransferases that employ nucleotide-linked sugars as their substrates. The focus of this report is GDP-perosamine synthase, a PLP-dependent enzyme that catalyzes the last step in the formation of GDP-perosamine, namely, the amination of the sugar C-4'. Here we describe the three-dimensional structure of the enzyme from Caulobacter crescentus determined to a nominal resolution of 1.8 A and refined to an R-factor of 17.9%. The overall fold of the enzyme places it into the well-characterized aspartate aminotransferase superfamily. Each subunit of the dimeric enzyme contains a seven-stranded mixed beta-sheet, a two-stranded antiparallel beta-sheet, and 12 alpha-helices. Amino acid residues from both subunits form the active sites of the GDP-perosamine synthase dimer. Recently, the structure of another PLP-dependent enzyme, GDP-4-keto-6-deoxy- d-mannose-3-dehydratase (or ColD), was determined in our laboratory, and this enzyme employs the same substrate as GDP-perosamine synthase. Unlike GDP-perosamine synthase, however, ColD functions as a dehydratase that removes the sugar C-3' hydroxyl group. By purifying the ColD product and reacting it with purified GDP-perosamine synthase, we have produced a novel GDP-linked sugar, GDP-4-amino-3,4,6-trideoxy- d-mannose. Details describing the X-ray structural investigation of GDP-perosamine synthase and the enzymatic synthesis of GDP-4-amino-3,4,6-trideoxy- d-mannose are presented.
 ...
PMID:GDP-perosamine synthase: structural analysis and production of a novel trideoxysugar. 1824 75

Perosamine (4-amino-4,6-dideoxy- d-mannose), or its N-acetylated form, is one of several dideoxy sugars found in the O-antigens of such infamous Gram-negative bacteria as Vibrio cholerae O1 and Escherichia coli O157:H7. It is added to the bacterial O-antigen via a nucleotide-linked version, namely GDP-perosamine. Three enzymes are required for the biosynthesis of GDP-perosamine starting from mannose 1-phosphate. The focus of this investigation is GDP-perosamine synthase from Caulobacter crescentus, which catalyzes the final step in GDP-perosamine synthesis, the conversion of GDP-4-keto-6-deoxymannose to GDP-perosamine. The enzyme is PLP-dependent and belongs to the aspartate aminotransferase superfamily. It contains the typically conserved active site lysine residue, which forms a Schiff base with the PLP cofactor. Two crystal structures were determined for this investigation: a site-directed mutant protein (K186A) complexed with GDP-perosamine and the wild-type enzyme complexed with an unnatural ligand, GDP-3-deoxyperosamine. These structures, determined to 1.6 and 1.7 A resolution, respectively, revealed the manner in which products, and presumably substrates, are accommodated within the active site pocket of GDP-perosamine synthase. Additional kinetic analyses using both the natural and unnatural substrates revealed that the K m for the unnatural substrate was unperturbed relative to that of the natural substrate, but the k cat was lowered by a factor of approximately 200. Taken together, these studies shed light on why GDP-perosamine synthase functions as an aminotransferase whereas another very similar PLP-dependent enzyme, GDP-4-keto-6-deoxy- d-mannose 3-dehydratase or ColD, catalyzes a dehydration reaction using the same substrate.
 ...
PMID:Accommodation of GDP-linked sugars in the active site of GDP-perosamine synthase. 1879 99

L-colitose and d-perosamine are unusual sugars found in the O-antigens of some Gram-negative bacteria such as Escherichia coli, Vibrio cholerae, and Salmonella enterica, among others. The biosynthetic pathways for these two sugars begin with the formation of GDP-mannose from d-mannose 1-phosphate and GTP followed by the subsequent dehydration and oxidation of GDP-mannose to yield GDP-4-keto-6-deoxymannose. Following the production of GDP-4-keto-6-deoxymannose, the two pathways diverge. In the case of GDP-perosamine biosynthesis, the next step involves an amination reaction at the C-4' position of the sugar, whereas in GDP-colitose production, the 3'-hydroxyl group is removed. The enzymes catalyzing these reactions are GDP-perosamine synthase and GDP-4-keto-6-deoxymannose-3-dehydratase (ColD), respectively. Both of these enzymes are pyridoxal 5'-phosphate (PLP) dependent, and their three-dimensional structures place them into the well-characterized aspartate aminotransferase superfamily. A comparison of the active site architecture of ColD from E. coli (strain 5a, type O55:H7) to that of GDP-perosamine synthase from Caulobacter crescentus CB15 suggested that only two mutations would be required to convert ColD into an aminotransferase. Here we present a combined structural and functional analysis of the ColD S187N/H188K mutant protein that, indeed, has been converted from a sugar dehydratase into an aminotransferase.
 ...
PMID:Two site-directed mutations are required for the conversion of a sugar dehydratase into an aminotransferase. 1940 12