STRUCTURE/FUNCTION OF PYRUVOYL AND PLP-DEPENDENT ENZYMES

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The structure-function relationships of basic amino acid decarboxylases will be studied by X-ray diffraction and protein engineering techniques. Most decarboxylases require pyridoxal phosphate (PLP) as an essential cofactor while others utilize a covalently bound a-keto acid. The proposed studies involve a decarboxylase of each cofactor type: a pyruvoyl- dependent histidine decarboxylase (Prv-HDC) and a PLP-dependent ornithine decarboxylase (PLP-ODC), both from Lactobacillus 30alpha, a Gram-positive bacterium resident in the mammalian gut. The corresponding mammalian enzymes are less amenable for X-ray diffraction studies. The decarboxylation of histidine is the only known mechanism for the biosynthesis of histamine. Physiological effects of histamine include vasodilation, hypotension, gastric HCl secretion, anaphylaxis, and mediation of the allergic response. The Prv-HDC from L. 30alpha is an inducible enzyme, and the most thoroughly studied of an unusual class of enzymes that produce their own prosthetic group from an internal amino acid residue. We have determined the X-ray structure of a low pH (hyperbolic kinetics) form of Prv-HDC and have proposed a mechanism of action on the basis of product and inhibitor complexes. The gene for HDC has been cloned in the laboratory of Dr. Jon Robertus and several site-specific mutants prepared. We have isolated and crystallized several such mutants for crystallographic study. In addition, we propose to produce a stable proHDC mutant (S82A) to study the activation properties of this enzyme and also examine a higher pH (sigmoidal kinetics) form of HDC. High resolution data of these systems will be collected with our Hamlin/Xuong area detector system and the structures refined on the University's Cray supercomputer. Ornithine decarboxylase (ODC) is a PLP-dependent enzyme that converts ornithine to putrescine which is a precursor of the polyamines spermine and spermidine. ODC is found in all cells but in particularly high activity in rapidly dividing cells. It has been used as a target for cancer chemotherapy and the inhibitor alpha-DL-difluoromethylornithine has been used successfully to treat Trypanosomal infections. The enzyme is generally noted for its rapid turnover rate with the mammalian enzyme having a stable half-life of only about 1 hour. In contrast, ODC from L. 30alpha is stable and representative of large subunit (80kDa) PLP decarboxylase. Our ODC crystals diffract to beyond 2.4A resolution and two potential heavy atom derivatives have been collected. We propose to determine the crystal structure of ODC, to clone and sequence the ODC gene, and later express it for site-directed mutagenesis studies on its active site.