Abstract
The biosynthesis of heme is strictly regulated, probably because of the toxic effects of excess heme and its biosynthetic precursors. In many organisms, heme biosynthesis starts with the production of 5-aminolevulinic acid (ALA) from glycine and succinyl-coenzyme A, a process catalyzed by a homodimeric enzyme, pyridoxal 5′-phosphate (PLP)-dependent 5-aminolevulinate synthase (ALAS). ALAS activity is negatively regulated by heme in various ways, such as the repression of ALAS gene expression, degradation of ALAS mRNA, and inhibition of mitochondrial translocation of the mammalian precursor protein. There has been no clear evidence, however, that heme directly binds to ALAS to negatively regulate its activity. We found that recombinant ALAS from Caulobacter crescentus was inactivated via a heme-mediated feedback manner, in which the essential coenzyme PLP was rel eased to form the inactive heme-bound enzyme. The spectroscopic properties of the heme-bound ALAS showed that a histidine-thiolate hexa-coordinated ferric heme bound to each subunit with a one-to-one stoichiometry. His340 and Cys398 were identified as the axial ligands of heme, and mutant ALASs lacking either of these ligands became resistant to heme-mediated inhibition. ALAS expressed in C. crescentus was also found to bind heme, suggesting that heme-mediated feedback inhibition of ALAS is physiologically relevant in C. crescentus.
Highlights
A number of studies on the catalytic mechanism of aminolevulinate synthase (ALAS) have been conducted[15,16], and crystal structures of ALASs from Rhodobacter capsulatus and Saccharomyces cerevisiae have been solved[17,18]
It is possible that aminolevulinic acid (ALA) may be formed via the C5 pathway in C. crescentus, the hemA gene encoding the first enzyme of the C5 pathway, glutamyl-tRNA reductase, has not been annotated in the C. crescentus genome or in the genome of any other bacteria classified into the α-proteobacteria group
The recombinant crescentus ALAS (cALAS) fused to a hexa-histidine-tag (His6-tag) at the C-terminus was successfully overexpressed in Escherichia coli
Summary
A number of studies on the catalytic mechanism of ALAS have been conducted[15,16], and crystal structures of ALASs from Rhodobacter capsulatus and Saccharomyces cerevisiae have been solved[17,18]. The C. crescentus genome database contains a gene (CC_3139; UniProt #Q9A3R3_CAUVC) encoding a protein with high similarity to glutamate-1-semialdehyde aminotransferase. This gene is annotated as hemL, the second gene of the C5 pathway of heme biosynthesis. We set out to characterize the enzymatic nature of cALAS, and during the course of our studies, we isolated an inactive heme-bound form of cALAS
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