Targeting the active site gate to yield hyperactive variants of 5-aminolevulinate synthase.
The rate of porphyrin biosynthesis in mammals is controlled by the activity of the pyridoxal 5'-phosphate-dependent enzyme 5-aminolevulinate synthase (EC 2.3.1.37). Based on the postulate that turnover in this enzyme is controlled by conformational dynamics associated with a highly conserved active site loop, we constructed a variant library by targeting imperfectly conserved noncatalytic loop residues and examined the effects on product and porphyrin production. Functional loop variants of the enzyme were isolated via genetic complementation in Escherichia coli strain HU227. Colony porphyrin fluorescence varied widely when bacterial cells harboring the loop variants were grown on inductive media; this 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 100-fold. Presteady state single turnover reaction data indicated that the second step of quinonoid intermediate decay, previously assigned as reaction rate-limiting, was specifically accelerated such that in three of the variants this step was no longer kinetically significant. Overall, our 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.
Lendrihas
Thomas
T
Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, Florida 33612, USA.
Hunter
Gregory A
GA
Ferreira
Gloria C
GC
D000624:5-Aminolevulinate Synthetase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000494:Allosteric Regulation / Q000502:physiology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005816:Genetic Complementation Test; D007700:Kinetics; D011166:Porphyrins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity / Q000502:physiology
D001426:Bacterial Proteins; D011166:Porphyrins; D011994:Recombinant Proteins; D000624:5-Aminolevulinate Synthetase