Linda D'Ari

p-Cresol formation by cell-free extracts of Clostridium difficile

Cell-free extracts of Clostridium difficile were shown to form p-cresol by decarboxylation of p-hydroxyphenylacetic acid. This activity required both high and low molecular weight fractions. The active component of the low molecular weight fraction had properties of an amino acid and could be replaced by serine, threonine or the corresponding alpha keto acids. Pyruvate was shown to function catalytically. Since the high molecular weight fraction was O2-sensitive and since dithionite was as effective as pyruvate with some high molecular weight fractions, the alpha keto acids probably serve as low potential reducing agents in this system. Because of instability, the p-cresolforming enzyme could not be purified.

[26] Synthesis of folylpolyglutamates

Publisher Summary This chapter provides an overview of the synthesis of folylpolyglutamates. The monoglutamate form of the folate coenzyme generally functions in place of the polyglutamyl forms. However, the affinity constants of the polyglutamyl forms of the coenzyme are from 10 to 1000 times less than that of the monoglutamyl form for some enzymes. The procedures that are described for the synthesis of the folylpolyglutamates include solid phase synthesis or procedures employing anhydrous solution chemistry for synthesis of the protected peptides of oligo-γ-glutamic acid. The overall approach to the synthesis of the pteroylpolyglutamates is presented in the chapter. The purity of the intermediates and the final products of the chemical syntheses is determined by the thin layer chromatography. The folylpolyglutamates are reduced to the 5,6,7,8-tetrahydro derivatives through the use of Lactobacillus casei dihydrofolate reductase at pH 5.5. This procedure gives about a 55% yield of (S)-isomer. Purification of the reduced product is carried out by chromatography on a QAE-Sephadex column.

Purification, crystallization, and preliminary x-ray studies of a bifunctional 5,10-methenyl/methylene-tetrahydrofolate cyclohydrolase/dehydrogenase from Escherichia coli.

A bifunctional enzyme that catalyzes the conversion of formyltetrahydrofolate to methylene‐tetrahydrofolate (5,10‐methenyltetrahydrofolate cyclohydrolase and 5,10‐methylene tetrahydrofolate dehydrogenease), has been subcloned from a cDNA library, purified to homogeneity, and crystallized. The crystals belong to space group I222, with unit cell dimensions of a= 64.5 Å b= 84.9 Å c= 146.1 Å. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme monomer, and a specific volume of the unit cell of 3.2 Å3/Da. The crystals diffract to at least 2.8 Å resolution after flash‐cooling, when using a rotating anode x‐ray source and an RAXIS image plate detector. A 2.56 Å resolution native data set has been collected at beamline X12‐C at the NSLS.

Purification, crystallization, and preliminary X‐ray studies of 10‐formyltetrahydrofolate synthetase from Clostridia acidici‐urici

The monofunctional enzyme 10‐formyltetrahydrofolate synthetase (THFS), which is responsible for the recruitment of single carbon units from the formate pool into a variety of folate‐dependent biosynthetic pathways, has been subcloned, purified, and crystallized. The crystals belong to space group P21, with unit cell dimensions a= 102.4 Å b= 116.5 Å c= 115.8 Å and β = 103.5 Å. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme tetramer, and a specific volume of the unit cell of 2.7 Å3/Da. The crystals diffract to at least 2.3 Å resolution after flash‐cooling, when using a rotating anode x‐ray source and an RAXIS image plate detector.