Pyridoxal 5-phosphate (PLP, vitamin B 6 ), 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 threecarbon phosphosugars to form PLP. In the 2.2-Å crystal structure, PdxS is a cylindrical dodecamer of subunits having the classic (␤/␣) 8 barrel fold. PdxS subunits form two hexameric rings with the active sites positioned on the inside. The hexamer and dodecamer forms coexist in solution. A novel phosphate-binding site is suggested by bound sulfate. The sulfate and another bound molecule, methyl pentanediol, were used to model the substrate ribulose 5-phosphate, and to propose catalytic roles for residues in the active site. The distribution of conserved surfaces in the PdxS dodecamer was used to predict a docking site for the glutaminase partner, PdxT.

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Low-molecular weight protein tyrosine phosphatases are virtually ubiquitous, which implies that they have important cellular functions. We present here the 2.2 A resolution X-ray crystallographic structure of wild-type LTP1, a low-molecular weight protein tyrosine phosphatase from Saccharomyces cerevisiae. We also present the structure of an inactive mutant substrate complex of LTP1 with p-nitrophenyl phosphate (pNPP) at a resolution of 1.7 A. The crystal structures of the wild-type protein and of the inactive mutant both have two molecules per asymmetric unit. The wild-type protein crystal was grown in HEPES buffer, a sulfonate anion that resembles the phosphate substrate, and a HEPES molecule was found with nearly full occupancy in the active site. Although the fold of LTP1 resembles that of its bovine counterpart BPTP, there are significant changes around the active site that explain differences in their kinetic behavior. In the crystal of the inactive mutant of LTP1, one molecule has a pNPP in the active site, while the other has a phosphate ion. The aromatic residues lining the walls of the active site cavity exhibit large relative movements between the two molecules. The phosphate groups present in the structures of the mutant protein bind more deeply in the active site (that is, closer to the position of nucleophilic cysteine side chain) than does the sulfonate group of the HEPES molecule in the wild-type structure. This further confirms the important role of the phosphate-binding loop in stabilizing the deep binding position of the phosphate group, thus helping to bring the phosphate close to the thiolate anion of nucleophilic cysteine, and facilitating the formation of the phosphoenzyme intermediate.

In bovine low Mr protein tyrosine phosphatase, the pKa values of His-66 and His-72 are 8.3 and 9.2, respectively. These unusually high values were hypothesized to be caused by electrostatic interactions with several nearby negatively charged groups. To test this, mutant enzymes were made in which one or more carboxylate side chains were removed or introduced near the histidines. Michaelis kinetic parameters, measured using p-nitrophenyl phosphate as a substrate, indicated that all mutant enzymes retained approximately 50% or more of the activity of wild-type enzyme. The effect that each mutation had on the pKa of the nearby histidine was monitored by 1H NMR spectroscopy using the MLEV-17 pulse sequence to filter out the broad interfering amide resonances in the spectra. Independently, computer simulations of the pKas were obtained using the finite difference method to solve the linear Poisson-Boltzmann equation. The proximity of a charged residue to the titrating histidine imidazole largely determined the extent of the pKa perturbation. The change in pKa for His-72 in the mutant enzymes was -1.69 units for D42A, -2.36 units for E23A, -2.99 units for E23A/D42A, and unchanged for E139A and Q143E. Thus, the pKa of His-72 in the double mutant E23A/D42A decreased to nearly that of a free histidine imidazole group. The His-66 pKa change was -1.25 units for E139A and was not significant for the other mutants. His-66, Glu-139, and Gln-143 are at the protein surface and much more exposed to the higher solvent dielectric compared to His-72, Glu-23, and Asp-42. These structural characteristics explain the smaller decrease in the observed pKa of His-66 for the E139A mutant compared to the decrease in the pKa of His-72 when a single nearby carboxylate was removed. These observations were adequately predicted by theoretical electrostatic calculations using the Poisson-Boltzmann equation as a model for a solute molecule of low dielectric in solution of high dielectric.