Abstract
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.
Highlights
Aminolevulinate (ALA)2 is the universal building block of tetrapyrolle biosynthesis [1]
The inferred conformational dynamics of this loop are of interest because kinetic and crystallographic studies support the hypothesis that the rate of ALA production is controlled by opening of the active site loop coincident with product release [6, 9, 10]
The porphyrin fluorescence in individual colonies differed substantially, as exemplified in Fig. 2, where bacterial cells harboring a hyperactive aminolevulinate synthase (ALAS) variant are compared with cells harboring the wild-type ALAS expression plasmid [21]
Summary
Materials—DEAE-Sephacel, -mercaptoethanol, PLP, bovine serum albumin, succinyl-CoA, ALA-hydrochloride, ␣-keto-glutarate, ␣-ketoglutarate dehydrogenase, Bis-Tris, HEPES-free acid, MOPS, thiamin pyrophosphate, NADϩ, and the bicinchoninic acid protein determination kit were purchased from Sigma-Aldrich. Presteady state kinetic reactions of the variant enzymes were examined under single turnover conditions, using final concentrations of 60 M enzyme, 120 mM glycine, and 10 M succinyl-CoA in 100 mM HEPES, pH 7.5, and 10% (v/v) glycerol as previously described [9]. The spectral data were used to determine the observed rate constants via the Robust Global Fitting program This approach utilizes the single value decomposition software provided by OLIS, Inc. as previously reported [9, 10]. The changes in fluorescence at 428 nm were plotted as a function of ALA concentration, and the KD value was determined by fitting the data to Equation 3, using nonlinear regression analysis software. The natural log of the calculated values for the turnover numbers (lnkcat) were plotted versus the inverse of temperature, and the data were fit to the Arrhenius equation
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