Dataset Information

Investigating the bifunctionality of cyclizing and "classical" 5-aminolevulinate synthases.

ABSTRACT: The precursor to all tetrapyrroles is 5-aminolevulinic acid, which is made either via the condensation of glycine and succinyl-CoA catalyzed by an ALA synthase (the C4 or Shemin pathway) or by a pathway that uses glutamyl-tRNA as a precursor and involves other enzymes (the C5 pathway). Certain ALA synthases also catalyze the cyclization of ALA-CoA to form 2-amino-3-hydroxycyclopent-2-en-1-one. Organisms with synthases that possess this second activity nevertheless rely upon the C5 pathway to supply ALA for tetrapyrrole biosynthesis. The C5 N units are components of a variety of secondary metabolites. Here, we show that an ALA synthase used exclusively for tetrapyrrole biosynthesis is also capable of catalyzing the cyclization reaction, albeit at much lower efficiency than the dedicated cyclases. Two absolutely conserved serines present in all known ALA-CoA cyclases are threonines in all known ALA synthases, suggesting they could be important in distinguishing the functions of these enzymes. We found that purified mutant proteins having single and double substitutions of the conserved residues are not improved in their respective alternate activities; rather, they are worse. Protein structural modeling and amino acid sequence alignments were explored within the context of what is known about the reaction mechanisms of these two different types of enzymes to consider what other features are important for the two activities.

SUBMITTER: Liu J

PROVIDER: S-EPMC5775165 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Investigating the bifunctionality of cyclizing and "classical" 5-aminolevulinate synthases.

Liu Joyce J Kaganjo James J Zhang Wenjun W Zeilstra-Ryalls Jill J

Protein science : a publication of the Protein Society 20171128 2

The precursor to all tetrapyrroles is 5-aminolevulinic acid, which is made either via the condensation of glycine and succinyl-CoA catalyzed by an ALA synthase (the C4 or Shemin pathway) or by a pathway that uses glutamyl-tRNA as a precursor and involves other enzymes (the C5 pathway). Certain ALA synthases also catalyze the cyclization of ALA-CoA to form 2-amino-3-hydroxycyclopent-2-en-1-one. Organisms with synthases that possess this second activity nevertheless rely upon the C5 pathway to sup ...[more]

PMID: 29027286

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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

Investigating the bifunctionality of cyclizing and "classical" 5-aminolevulinate synthases.

Publications

Investigating the bifunctionality of cyclizing and "classical" 5-aminolevulinate synthases.

Similar Datasets

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