Crystal structures capture three states in the catalytic cycle of a pyridoxal phosphate (PLP) synthase.

PLP synthase (PLPS) is a remarkable single-enzyme biosynthetic pathway that produces pyridoxal 5'-phosphate (PLP) from glutamine, ribose 5-phosphate, and glyceraldehyde 3-phosphate. The intact enzyme includes 12 synthase and 12 glutaminase subunits. PLP synthesis occurs in the synthase active site by a complicated mechanism involving at least two covalent intermediates at a catalytic lysine.

Heterologous production, purification and characterization of enzymatically active Sindbis virus nonstructural protein nsP1.

Alphavirus nonstructural protein nsP1 possesses distinct methyltransferase (MTase) and guanylyltransferase (GTase) activities involved in the capping of viral RNAs. In alphaviruses, the methylation of GTP occurs before RNA transguanylation and nsP1 forms a covalent complex with m(7)GMP unlike the host mRNA guanylyltransferase which forms GMP-enzyme complex. In this study, full length SINV nsP1 was expressed in a soluble form with an N-terminal histidine tag in Escherichia coli and purified to homogeneity.

A new arrangement of (beta/alpha)8 barrels in the synthase subunit of PLP synthase.

Pyridoxal 5'-phosphate (PLP, vitamin B6), a cofactor in many enzymatic reactions, has two distinct biosynthetic routes, which do not coexist in any organism. Two proteins, known as PdxS and PdxT, together form a PLP synthase in plants, fungi, archaea, and some eubacteria. PLP synthase is a heteromeric glutamine amidotransferase in which PdxT produces ammonia from glutamine and PdxS combines ammonia with five- and three-carbon phosphosugars to form PLP.

Kinetic and spectroscopic studies of Tritrichomonas foetus low-molecular weight phosphotyrosyl phosphatase. Hydrogen bond networks and electrostatic effects.

Virtually all of the eukaryotic low-molecular weight protein tyrosine phosphatases (LMW PTPases) studied to date contain a conserved, high-pK(a) histidine residue that is hydrogen bonded to a conserved active site asparagine residue of the phosphate binding loop. However, in the putative enzyme encoded by the genome of the trichomonad parasite Tritrichomonas foetus, this otherwise highly conserved histidine is replaced with a glutamine residue. We have cloned the gene, expressed the enzyme, demonstrated its catalytic activity, and examined the structural and functional roles of the glutamine residue using site-directed mutagenesis, kinetic measurements, and NMR spectroscopy.