An Extended C-Terminus, the Possible Culprit for Differential Regulation of 5-Aminolevulinate Synthase Isoforms

Abstract

5-Aminolevulinate synthase (ALAS; E.C. 2.3.1.37) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the key regulatory step of porphyrin biosynthesis in metazoa, fungi, and α-proteobacteria. ALAS is evolutionarily related to transaminases and is therefore classified as a fold type I PLP-dependent enzyme. As an enzyme controlling the key committed and rate-determining step of a crucial biochemical pathway ALAS is ideally positioned to be subject to allosteric feedback inhibition. Extensive kinetic and mutational studies demonstrated that the overall enzyme reaction is limited by subtle conformational changes of a hairpin loop gating the active site. These findings, coupled with structural information, facilitated early prediction of allosteric regulation of activity via an extended C-terminal tail unique to eukaryotic forms of the enzyme. This prediction was subsequently supported by the discoveries that mutations in the extended C-terminus of the erythroid ALAS isoform (ALAS2) cause a metabolic disorder known as X-linked protoporphyria not by diminishing activity, but by enhancing it. Furthermore, kinetic, structural, and molecular modeling studies demonstrated that the extended C-terminal tail controls the catalytic rate by modulating conformational flexibility of the active site loop. However, the precise identity of any such molecule remains to be defined. Here we discuss the most plausible allosteric regulators of ALAS activity based on divergences in AlphaFold-predicted ALAS structures and suggest how the mystery of the mechanism whereby the extended C-terminus of mammalian ALASs allosterically controls the rate of porphyrin biosynthesis might be unraveled.

Elucidating the Role of Human ALAS2 C-terminal Mutations Resulting in Loss of Function and Disease

“…The predicted pathogenicity of the other variants was either ambiguous or benign, including V562A. Thus, computational analysis of the hALAS2 C-terminus alone did not confidently yield insight into a potential molecular basis for enzyme dysfunction as it is probable that the extended C-terminus adopts an ensemble of biologically relevant conformations …”

“…Thus, computational analysis of the hALAS2 C-terminus alone did not confidently yield insight into a potential molecular basis for enzyme dysfunction as it is probable that the extended C-terminus adopts an ensemble of biologically relevant conformations. 51 Hydrophobic C-terminal Mutations Differentially Impact In Vitro Protein Stability. A previous report identified that both V562A and M567I had significantly different stability in HEK293 cells, in which hALAS2 V562A had a shorter half-life than WT, and M567I was significantly stabilized compared to WT.…”

Cimetidine Does Not Inhibit 5-Aminolevulinic Acid Synthase or Heme Oxygenase Activity: Implications for Treatment of Acute Intermittent Porphyria and Erythropoietic Protoporphyria

“…Not only do ALAS1 and ALAS2 catalyze the same reaction but they also have highly similar gene structures and are believed to have emerged from gene duplication. The catalytic cores of the two human enzymes are 75% identical in amino acid sequence [55] and are the same length. In line with our findings, the structure of cimetidine has limited similarity to the ALAS substrates, glycine and succinyl-CoA, as well as to ALA and other heme intermediates (Supplemental Figure S2).…”

An adaptor for feedback regulation of heme biosynthesis by the mitochondrial protease CLPXP

“…Several alleles of ALAS2 that truncate the C-terminal extension lead to a form of erythropoietic protoporphyria (XLDPP) (29,30). Although this disorder is likely due in part to the several-fold increase in enzyme activity attributed to loss of autoinhibition by the C-terminus (14,15,30,31), a C-terminally truncated ALAS2 variant also exhibited reduced turnover in cells (32). We propose that loss of degradation by CLPXP exacerbates the excess of ALAS2 activity in XLDPP.…”