DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, DNA is visualized by staining. This article demonstrates how to use thiazole orange to stain DNA. Thiazole orange compares favorably to common staining methods, in that it is sensitive, inexpensive, excitable with UV or blue light (to prevent sample damage), and safer than ethidium bromide. Labs already equipped to run DNA electrophoresis experiments using ethidium bromide can generally switch dyes with no additional changes to existing protocols, using UV light for detection. Blue-light detection to avoid sample damage can additionally be achieved with a blue-light source and emission filter. Labs already equipped for blue-light detection can simply switch dyes with no additional changes to existing protocols.
The flavoenzyme UDP-galactopyranose mutase (UGM) is a mediator of cell wall biosynthesis in many pathogenic microorganisms. UGM catalyzes a unique ring contraction reaction that results in the conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). UDP-Galf is an essential precursor to the galactofuranose residues found in many different cell wall glycoconjugates. Due to the important consequences of UGM catalysis, structural and biochemical studies are needed to elucidate the mechanism and identify the key residues involved. Here, we report the results of site-directed mutagenesis studies on the absolutely conserved residues in the putative active site cleft. By generating variants of the UGM from Klebsiella pneumoniae, we have identified two arginine residues that play critical catalytic roles (alanine substitution abolishes detectable activity). These residues also have a profound effect on the binding of a fluorescent UDP derivative that inhibits UGM, suggesting that the Arg variants are defective in their ability to bind substrate. One of the residues, Arg280, is located in the putative active site, but, surprisingly, the structural studies conducted to date suggest that Arg174 is not. Molecular dynamics simulations indicate that closed UGM conformations can be accessed in which this residue contacts the pyrophosphoryl group of the UDP-Gal substrates. These results provide strong evidence that the mobile loop, noted in all the reported crystal structures, must move in order for UGM to bind its UDP-galactose substrate.
Galactofuranose residues are key components of the cell walls of numerous pathogenic bacteria. UDP‐galactofuranose (UDP‐Gal f ), a biosynthetic precursor of Gal f containing glycoconjugates, is produced from UDP‐galactopyranose (UDP‐Gal p ) by the flavoenzyme UDP‐galactopyranose mutase (UGM). We have accumulated evidence that the enzymatic reaction proceeds through an iminium intermediate formed between flavin and the substrate. To understand this unique mode of catalysis, we determined the ligand‐bound structure of UGM. We report the structure of UGM from Klebsiella pneumoniae bound to the substrate analog UDP‐glucose (UDP‐Glc) at 2.45 Å resolution, in which the crystallographic asymmetric unit contains the UGM dimer in distinct closed and open modes. In the closed monomer, a short loop restructures into an α‐helix, orienting a conserved arginine residue toward the phosphoryl groups of the nucleotide sugar, and positioning the Glc moiety near the flavin cofactor. The uracil moiety stacks with a conserved tyrosine residue, rather than the previously proposed Trp160. Despite its similarity to the natural substrate, UDP‐Glc does not participate in iminium formation upon binding to the enzyme. This observation can be rationalized by the structure; the glucose ring of the nucleotide sugar appears to be oriented improperly for catalysis. From the structure, we propose a model for the UGM‐substrate complex. Supported by NIH AI063596
The flavoenzyme uridine 5′-diphosphate galactopyranose mutase (UGM or Glf) catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose. The latter is a key building block for cell wall construction in numerous pathogens, including Mycobacterium tuberculosis. Mechanistic studies of UGM suggested a novel role for the flavin, and we previously provided evidence that the catalytic mechanism proceeds through a covalent flavin−galactose iminium. Here, we describe 2.3 and 2.5 Å resolution X-ray crystal structures of the substrate-bound enzyme in oxidized and reduced forms, respectively. In the latter, C1 of the substrate is 3.6 Å from the nucleophilic flavin N5 position. This orientation is consistent with covalent catalysis by flavin.
