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. Aminolevulinate (ALA)2 is the universal building block of tetrapyrolle biosynthesis (1). In nonplant eukaryotes and the ␣-subclass of purple bacteria, the production of ALA is catalyzed by the pyridoxal 5Ј-phosphate (PLP)-dependent enzyme 5-aminolevulinate synthase (ALAS) (EC 2.3.1.37), in a reaction involving the Claisen-like condensation of succinyl-coenzyme-A and glycine to yield CoA, carbon dioxide, and ALA (2). ALAS catalyzes the first committed step of tetrapyrrole biosynthesis in these organisms, which is also the rate-determining step of the pathway. Consequently, overexpression of ALAS in prokaryotic and eukaryotic cells results in accumulation of the photosensitizing heme precursor protoporphyrin IX (3). This property could potentially lead to novel applications of ALAS or ALAS variants in photodynamic therapy of tumors and other dysplasias (4).ALAS is classified as a fold-type I PLP-dependent enzyme and, like the evolutionarily related L-amino acid transaminases (5), functions as a homodimer wherein a PLP cofactor is bound at each of the two active sites, which are recessed in clefts at the subunit interface (6, 7). X-ray crystal structures of ALAS from Rhodobacter capsulatus and the closely related enzyme 8-amino-7-oxononanoate synthase from Escherichia coli reveal an induced fit type mechanism wherein binding of substrates and product, respectively, trigger closure of an extended loop over the active site (6, 8) (Fig. 1). The inferred conformational dynamics of this loop are of interest because kinetic and crystallographic studies support the hypothes...

5-Aminolevulinate synthase (EC 2.3.1.37) (ALAS), a pyridoxal 5-phosphate (PLP)-dependent enzyme, catalyzes the initial step of heme biosynthesis in animals, fungi, and some bacteria. Condensation of glycine and succinyl coenzyme A produces 5-aminolevulinate, coenzyme A, and carbon dioxide. X-ray crystal structures of Rhodobacter capsulatus ALAS reveal that a conserved active site serine moves to within hydrogen bonding distance of the phenolic oxygen of the PLP cofactor in the closed substrate-bound enzyme conformation and within 3-4 Å of the thioester sulfur atom of bound succinyl-CoA. To evaluate the role(s) of this residue in enzymatic activity, the equivalent serine in murine erythroid ALAS was substituted with alanine or threonine. Although both the K m SCoA and k cat values of the S254A variant increased, by 25-and 2-fold, respectively, the S254T substitution decreased k cat without altering K m SCoA . Furthermore, in relation to wild-type ALAS, the catalytic efficiency of S254A toward glycine improved ϳ3-fold, whereas that of S254T diminished ϳ3-fold. Circular dichroism spectroscopy revealed that removal of the side chain hydroxyl group in the S254A variant altered the microenvironment of the PLP cofactor and hindered succinyl-CoA binding. Transient kinetic analyses of the variant-catalyzed reactions and protein fluorescence quenching upon 5-aminolevulinate binding demonstrated that the protein conformational transition step associated with product release was predominantly affected. We propose the following: 1) Ser-254 is critical for formation of a competent catalytic complex by coupling succinyl-CoA binding to enzyme conformational equilibria, and 2) the role of the active site serine should be extended to the entire ␣-oxoamine synthase family of PLP-dependent enzymes.

The first enzyme of haem biosynthesis, ALAS (5-aminolaevulinic acid synthase), catalyses the pyridoxal 5'-phosphate-dependent condensation of glycine and succinyl-CoA to 5-aminolaevulinic acid, CO(2) and CoA. The crystal structure of Rhodobacter capsulatus ALAS provides the first snapshots of the structural basis for substrate binding and catalysis. To elucidate the functional role of single amino acid residues in the active site for substrate discrimination, substrate positioning, catalysis and structural protein rearrangements, multiple ALAS variants were generated. The quinonoid intermediates I and II were visualized in single turnover experiments, indicating the presence of an α-amino-β-oxoadipate intermediate. Further evidence was obtained by the pH-dependent formation of quinonoid II from the product 5-aminolaevulinic acid. The function of Arg(21), Thr(83), Asn(85) and Ile(86), all involved in the co-ordination of the succinyl-CoA substrate carboxy group, were analysed kinetically. Arg(21), Thr(83)and Ile(86), all of which are located in the second subunit to the intersubunit active site, were found to be essential. Their location in the second subunit provides the basis for the required structural dynamics during the complex condensation of both substrates. Utilization of L-alanine by the ALAS variant T83S indicated the importance of this residue for the selectiveness of binding with the glycine substrate compared with related amino acids. Asn(85) was found to be solely important for succinyl-CoA substrate recognition and selectiveness of binding. The results of the present study provide a novel dynamic view on the structural basis of ALAS substrate-binding and catalysis.

5-Aminolevulinate synthase (ALAS) controls the rate-limiting step of heme biosynthesis in mammals by catalyzing the condensation of succinyl-coenzyme A and glycine to produce 5-aminolevulinate, coenzyme-A (CoA), and carbon dioxide. ALAS is a member of the a-oxoamine synthase family of pyridoxal 5 0 -phosphate (PLP)-dependent enzymes and shares high degree of structural similarity and reaction mechanism with the other members of the family. The X-ray crystal structure of ALAS from Rhodobacter capsulatus reveals that the alkanoate component of succinyl-CoA is coordinated by a conserved arginine and a threonine. The functions of the corresponding acyl-CoA-binding residues in murine erthyroid ALAS (R85 and T430) in relation to acyl-CoA binding and substrate discrimination were examined using site-directed mutagenesis and a series of CoA-derivatives. The catalytic efficiency of the R85L variant with octanoyl-CoA was 66-fold higher than that of the wild-type protein, supporting the proposal of this residue as key in discriminating substrate binding. Substitution of the acyl-CoA-binding residues with hydrophobic amino acids caused a ligand-induced negative dichroic band at 420 nm in the CD spectra, suggesting that these residues affect substrate-mediated changes to the PLP microenvironment. Transient kinetic analyses of the R85K variant-catalyzed reactions confirm that this substitution decreases microscopic rates associated with formation and decay of a key reaction intermediate and show that the nature of the acyl-CoA tail seriously affect product binding. These results show that the bifurcate interaction of the carboxylate moiety of succinyl-CoA with R85 and T430 is an important determinant in ALAS function and may play a role in substrate specificity.