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Circular permutation of 5-aminolevulinate synthase. Mapping the polypeptide chain to its function.

ABSTRACT: 5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and some prokaryotes. The enzyme functions as a homodimer and requires pyridoxal 5'-phosphate as a cofactor. Although the roles of defined amino acids in the active site and catalytic mechanism have been recently explored using site-directed mutagenesis, much less is known about the role of the 5-aminolevulinate synthase polypeptide chain arrangement in folding, structure, and ultimately, function. To assess the importance of the continuity of the polypeptide chain, circularly permuted 5-aminolevulinate synthase variants were constructed through either rational design or screening of an engineered random library. One percent of the random library clones were active, and a total of 21 active variants had sequences different from that of the wild type 5-aminolevulinate synthase. Out of these 21 variants, 9 displayed unique circular permutations of the 5-aminolevulinate synthase polypeptide chain. The new termini of the active variants disrupted secondary structure elements and loop regions and fell in 100 amino acid regions from each terminus. This indicates that the natural continuity of the 5-aminolevulinate synthase polypeptide chain and the sequential arrangement of the secondary structure elements are not requirements for proper folding, binding of the cofactor, or assembly of the two subunits. Furthermore, the order of two identified functional elements (i.e. the catalytic and the glycine-binding domains) is apparently irrelevant for proper functioning of the enzyme. Although the wild type 5-aminolevulinate synthase and the circularly permuted variants appear to have similar, predicted overall tertiary structures, they exhibit differences in the arrangement of the secondary structure elements and in the cofactor-binding site environment. Taken together, the data lead us to propose that the 5-aminolevulinate synthase overall structure can be reached through multiple or alternative folding pathways.

SUBMITTER: Cheltsov AV

PROVIDER: S-EPMC4547487 | biostudies-literature | 2001 Jun

REPOSITORIES: biostudies-literature

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Circular permutation of 5-aminolevulinate synthase. Mapping the polypeptide chain to its function.

Cheltsov A V AV Barber M J MJ Ferreira G C GC

The Journal of biological chemistry 20010315 22

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and some prokaryotes. The enzyme functions as a homodimer and requires pyridoxal 5'-phosphate as a cofactor. Although the roles of defined amino acids in the active site and catalytic mechanism have been recently explored using site-directed mutagenesis, much less is known about the role of the 5-aminolevulinate synthase polypeptide chain arrangement in folding, structure, and ultimately, func ...[more]

PMID: 11279050

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Project description:5-Aminolevulinate (ALA) synthase (ALAS), a homodimeric pyridoxal-5'-phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in metazoa, fungi and α-proteobacteria. In this review, we focus on the advances made in unraveling the mechanism of the ALAS-catalyzed reaction during the past decade. The interplay between the PLP cofactor and the protein moiety determines and modulates the multi-intermediate reaction cycle of ALAS, which involves the decarboxylative condensation of two substrates, glycine and succinyl-CoA. Substrate binding and catalysis are rapid, and product (ALA) release dominates the overall ALAS kinetic mechanism. Interconversion between a catalytically incompetent, open conformation and a catalytically competent, closed conformation is linked to ALAS catalysis. Reversion to the open conformation, coincident with ALA dissociation, defines the slowest step of the reaction cycle. These findings were further substantiated by introducing seven mutations in the16-amino acid loop that gates the active site, yielding an ALAS variant with a greatly increased rate of catalytic turnover and heightened specificity constants for both substrates. Recently, molecular dynamics (MD) simulation analysis of various dimeric ALAS forms revealed that the seven active site loop mutations caused the proteins to adopt different conformations. In particular, the emergence of a β-strand in the mutated loop, which interacted with two preexisting β-strands to form an anti-parallel three-stranded β-sheet, conferred the murine heptavariant with a more stable open conformation and prompted faster product release than wild-type mALAS2. Moreover, the dynamics of the mALAS2 active site loop anti-correlated with that of the 35 amino acid C-terminal sequence. This led us to propose that this C-terminal extension, which is absent in prokaryotic ALASs, finely tunes mammalian ALAS activity. Based on the above results, we extend our previous proposal to include that discovery of a ligand inducing the mammalian C-terminal extension to fold offers a good prospect for the development of a new drug for X-linked protoporphyria and/or other porphyrias associated with enhanced ALAS activity and/or porphyrin accumulation.

Dataset Information

Circular permutation of 5-aminolevulinate synthase. Mapping the polypeptide chain to its function.

Publications

Circular permutation of 5-aminolevulinate synthase. Mapping the polypeptide chain to its function.

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