This 2-step assay provides a means to classify MS patients as having detectable or not detectable levels of anti-JCV antibodies. The finding that all 17 of the pre-PML samples that were available tested seropositive, and none tested seronegative, warrants further research on the clinical utility of the anti-JCV antibody assay as a potential tool for stratifying MS patients for higher or lower risk of developing PML.
Pyridoxal 5'-phosphate (PLP) is the biologically active form of vitamin B 6 and is an important cofactor for several of the enzymes involved in the metabolism of amine-containing natural products such as amino acids and amino-sugars. The PLP synthase holoenzyme consists of two subunits: YaaD catalyzes the condensation of ribulose 5-phosphate, glyceraldehyde-3-phosphate and ammonia and YaaE catalyzes the production of ammonia from glutamine. Here we describe the structure of the PLP synthase complex (YaaD-YaaE) from Thermotoga maritima at 2.9 Å resolution. This complex consists of a core of 12 YaaD monomers with 12 noninteracting YaaE monomers attached to the core. Compared to the previously published structure of PdxS (a YaaD ortholog in Geobacillus stearothermophilus), the N-terminus (1-18), which includes helix α0, the β2-α2 loop(46-56), which includes new helix α2a, and the C-terminus (270-280) of YaaD, are ordered in the complex but disordered in PdxS. A ribulose 5-phosphate is bound to YaaD via an imine with Lys82. Previous studies have demonstrated a similar imine at Lys149 and not at Lys81 (equivalent to Lys150 and 82 in T. maritima) for the Bacillus subtilis enzyme suggesting the possibility that two separate sites on YaaD are involved in PLP formation. A phosphate from the crystallization solution is found bound to YaaD and also serves as a marker for a possible second active site. An ammonia channel that connects the active site of YaaE with the ribulose 5-phosphate binding site was identified. This channel is similar to one found in imidazole glycerol phosphate synthase; however, when the β-barrels of the two complexes are superimposed, the glutaminase domains are rotated by about 180°w ith respect to each other.Pyridoxal 5'-phosphate (4, PLP) is the biologically active form of vitamin B 6 and is an important cofactor for several of the enzymes involved in the metabolism of amine-containing natural products such as amino acids and amino-sugars (1-4). There are two distinct PLP biosynthetic pathways that have not yet been found to coexist in the same organism (5). The Escherichia coli pathway has been extensively studied (6)(7)(8)(9)(10)(11)(12). In this pathway, PdxJ catalyzes the formation of pyridoxine 5'-phosphate (3, PNP) from 3-phosphohydroxy-1-aminoacetone 2 and 1-deoxy-D-xyulose 5-phosphate 1. This is then oxidized to PLP 4 by PdxH (Scheme 1).In the alternative pathway, PLP is formed from ribose 5-phosphate (5, R5P), glyceraldehyde 3-phosphate (6, G3P) and ammonia formed by the hydrolysis of glutamine (13-18) (Scheme 1
METHODS AND MATERIALS
Molecular CloningStandard methods were used for DNA restriction endonuclease digestion, ligation and transformation of DNA. Genomic DNA and plasmid DNA were purified with a Wizard Plus SV genomic DNA kit and a DNA Miniprep kit (Promega), respectively. DNA fragments were separated by agarose gel electrophoresis, excised and purified with the QiaExII (Qiagen). E. coli strain DH5 α was used as a recipient for transformation during plasmid construction ...
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