Hyperactive Variants of 5-aminolevulinate Synthase and Methods of Use
The rate of porphyrin biosynthesis in mammals is controlled by the activity of the pyridoxal 5'-phosphate-dependent enzyme 5-aminolevulinate synthase. Assuming the turnover in this enzyme is controlled by conformational dynamics at a highly conserved active site loop, a variant library was constructed by targeting imperfectly conserved non-catalytic loop residues and the effects on product and porphyrin production were examined. Functional loop variants of the enzyme were tested for porphyrin fluorescence, which varied widely and thus facilitated identification of clones encoding unusually active enzyme variants. Nine loop variants leading to high in vivo porphyrin production were purified and characterized kinetically. Steady-state catalytic efficiencies for the two substrates were increased by up to one hundred-fold. The data support the postulate that the active site loop controls the rate of product and porphyrin production in vivo and suggest the possibility of an as yet undiscovered means of allosteric regulation.
HUNTER GREGORY A [US]; FERREIRA GLORIA C [US]; LENDRIHAS THOMAS [US]
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