David H. Calhoun

The Correct Phylogenetic Relationship of KdsA (3-Deoxy-D-manno-octulosonate 8-Phosphate Synthase) with One of Two Independently Evolved Classes of AroA (3-Deoxy-D-arabino-heptulosonate 7-Phosphate Synthase)

-manno-octulosonate8-phosphate (KDOP) synthases.DAHP is the initial product that is specifically com-mitted to the biosynthesis of aromatic amino acids and avariety of other aromatic compounds via the action ofDAHP synthase. KDOP is best known as a key precursorof lipopolysaccharide in gram-negative bacteria, but itswider distribution in capsular polysaccharides, lipogly-can of Chlorella, and cell walls of higher plants impliesother functional roles at the cell surface as well [seeBrabetz et al. (2000) and references therein]. In 1996Walker et al. reported the existence of a class of DAHPsynthase (their Class II) that lacked observable homol-ogy with the then-known DAHP synthases (their ClassI). Class II DAHP synthases were described as 54,000-M

Inhibition of recombinant Pneumocystis carinii dihydropteroate synthetase by sulfa drugs.

Forty-four sulfa drugs were screened against crude preparations of recombinant Pneumocystis carinii dihydropteroate synthetase. The apparent Michaelis-Menten constants (Km) for p-aminobenzoic acid and 7,8-dihydro-6-hydroxymethylpterin pyrophosphate were 0.34 +/- 0.02 and 2.50 +/- 0.71 microM, respectively. Several sulfa drugs, including sulfathiazole, sulfachlorpyridazine, sulfamethoxypyridazine, and sulfathiourea, inhibited dihydropteroate synthetase approximately as well as sulfamethoxazole, as determined by the concentrations which cause 50% inhibition and/or by Ki. For all sulfones and sulfonamides tested, unsubstituted p-amino groups were necessary for activity, and sulfonamides containing an N1-heterocyclic substituent were found to be the most effective inhibitors. Folate biosynthesis in isolated intact P. carinii was approximately equally sensitive to inhibition by sulfamethoxazole, sulfachlorpyridazine, sulfamethoxypyridazine, sulfisoxazole, and sulfathiazole. Two of these drugs, sulfamethoxypyridazine and sulfisoxazole, are known to be less toxic than sulfamethoxazole and should be further evaluated for the treatment of P. carinii pneumonia.

Carboxyl-terminal truncations alter the activity of the human α-galactosidase A.

Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the human lysosomal enzyme, α-galactosidase A (αGal), leading to strokes, myocardial infarctions, and terminal renal failure, often leading to death in the fourth or fifth decade of life. The enzyme is responsible for the hydrolysis of terminal α-galactoside linkages in various glycolipids. Enzyme replacement therapy (ERT) has been approved for the treatment of Fabry disease, but adverse reactions, including immune reactions, make it desirable to generate improved methods for ERT. One approach to circumvent these adverse reactions is the development of derivatives of the enzyme with more activity per mg. It was previously reported that carboxyl-terminal deletions of 2 to 10 amino acids led to increased activity of about 2 to 6-fold. However, this data was qualitative or semi-quantitative and relied on comparison of the amounts of mRNA present in Northern blots with αGal enzyme activity using a transient expression system in COS-1 cells. Here we follow up on this report by constructing and purifying mutant enzymes with deletions of 2, 4, 6, 8, and 10 C-terminal amino acids (Δ2, Δ4, Δ6, Δ8, Δ10) for unambiguous quantitative enzyme assays. The results reported here show that the k cat/K m approximately doubles with deletions of 2, 4, 6 and 10 amino acids (0.8 to 1.7-fold effect) while a deletion of 8 amino acids decreases the k cat/K m (7.2-fold effect). These results indicate that the mutated enzymes with increased activity constructed here would be expected to have a greater therapeutic effect on a per mg basis, and could therefore reduce the likelihood of adverse infusion related reactions in Fabry patients receiving ERT treatment. These results also illustrate the principle that in vitro mutagenesis can be used to generate αGal derivatives with improved enzyme activity.