Dataset Information

Functional asymmetry for the active sites of linked 5-aminolevulinate synthase and 8-amino-7-oxononanoate synthase.

ABSTRACT: 5-Aminolevulinate synthase (ALAS) and 8-amino-7-oxononanoate synthase (AONS) are homodimeric members of the ?-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes. Previously, linking two ALAS subunits into a single polypeptide chain dimer yielded an enzyme (ALAS/ALAS) with a significantly greater turnover number than that of wild-type ALAS. To examine the contribution of each active site to the enzymatic activity of ALAS/ALAS, the catalytic lysine, which also covalently binds the PLP cofactor, was substituted with alanine in one of the active sites. Albeit the chemical rate for the pre-steady-state burst of ALA formation was identical in both active sites of ALAS/ALAS, the k(cat) values of the variants differed significantly (4.4±0.2 vs. 21.6±0.7 min(-1)) depending on which of the two active sites harbored the mutation. We propose that the functional asymmetry for the active sites of ALAS/ALAS stems from linking the enzyme subunits and the introduced intermolecular strain alters the protein conformational flexibility and rates of product release. Moreover, active site functional asymmetry extends to chimeric ALAS/AONS proteins, which while having a different oligomeric state, exhibit different rates of product release from the two ALAS and two AONS active sites due to the created intermolecular strain.

SUBMITTER: Turbeville TD

PROVIDER: S-EPMC3136873 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Functional asymmetry for the active sites of linked 5-aminolevulinate synthase and 8-amino-7-oxononanoate synthase.

Turbeville Tracy D TD Zhang Junshun J Adams W Christopher WC Hunter Gregory A GA Ferreira Gloria C GC

Archives of biochemistry and biophysics 20110511 1-2

5-Aminolevulinate synthase (ALAS) and 8-amino-7-oxononanoate synthase (AONS) are homodimeric members of the α-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes. Previously, linking two ALAS subunits into a single polypeptide chain dimer yielded an enzyme (ALAS/ALAS) with a significantly greater turnover number than that of wild-type ALAS. To examine the contribution of each active site to the enzymatic activity of ALAS/ALAS, the catalytic lysine, which also covalently bi ...[more]

PMID: 21600186

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Project description:Biosynthesis of heme represents a complex process that involves multiple stages controlled by different enzymes. The first of these proteins is a pyridoxal 5?-phosphate (PLP)-dependent homodimeric enzyme, 5-aminolevulinate synthase (ALAS), that catalyzes the rate-limiting step in heme biosynthesis, the condensation of glycine with succinyl-CoA. Genetic mutations in human erythroid-specific ALAS (ALAS2) are associated with two inherited blood disorders, X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP). XLSA is caused by diminished ALAS2 activity leading to decreased ALA and heme syntheses and ultimately ineffective erythropoiesis, whereas XLPP results from “gain-of-function” ALAS2 mutations and consequent overproduction of protoporphyrin IX and increase in Zn2+-protoporphyrin levels. All XLPP-linked mutations affect the intrinsically disordered C-terminal tail of ALAS2. Our earlier molecular dynamics (MD) simulation-based analysis showed that the activity of ALAS2 could be regulated by the conformational flexibility of the active site loop whose structural features and dynamics could be changed due to mutations. We also revealed that the dynamic behavior of the two protomers of the ALAS2 dimer differed. However, how the structural dynamics of ALAS2 active site loop and C-terminal tail dynamics are related to each other and contribute to the homodimer asymmetry remained unanswered questions. In this study, we used bioinformatics and computational biology tools to evaluate the role(s) of the C-terminal tail dynamics in the structure and conformational dynamics of the murine ALAS2 homodimer active site loop. To assess the structural correlation between these two regions, we analyzed their structural displacements and determined their degree of correlation. Here, we report that the dynamics of ALAS2 active site loop is anti-correlated with the dynamics of the C-terminal tail and that this anti-correlation can represent a molecular basis for the functional and dynamic asymmetry of the ALAS2 homodimer.

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

Functional asymmetry for the active sites of linked 5-aminolevulinate synthase and 8-amino-7-oxononanoate synthase.

Publications

Functional asymmetry for the active sites of linked 5-aminolevulinate synthase and 8-amino-7-oxononanoate synthase.

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