# Macromolecular interactions controlling the ALA synthases, keystone enzymes that initiate heme biosynthesis

> **NIH NIH R01** · MASSACHUSETTS INSTITUTE OF TECHNOLOGY · 2020 · $418,367

## Abstract

Heme is the oxygen-binding ligand of hemoglobin and is an essential cofactor or sensor element in many
proteins. Heme production must be tightly controlled to adequately supply these functions but to avoid
overproduction, as accumulation of free heme and heme precursors is toxic. The first committed step in heme
biosynthesis is the condensation of glycine and succinyl-CoA to yield 5-aminolevulinic acid (ALA). This reaction
is catalyzed by ALA synthase (ALAS), which uses pyridoxal 5ʹ-phosphate (PLP, the active form of vitamin B6)
as an essential cofactor. In animals, there are two differentially expressed ALAS isoforms. ALAS1 is present in
most cells, whereas ALAS2 is an erythroid-specific enzyme that is dramatically upregulated during red cell
development. In humans, mutations in ALAS2 cause two diseases: (1) X-linked sideroblastic anemia (XLSA)
when enzyme activity is too low to support healthy levels of heme production and erythropoiesis and (2)
Erythroid X-linked protoporphyria (XLPP), from gain-of-function ALAS2 mutations that overproduce ALA,
causing build up of toxic heme biosynthetic intermediates. The life cycle of ALAS is tightly regulated at steps
including mitochondrial import and protein turnover. Both these steps are feedback controlled by heme-binding.
Enzyme activity (and/or stability) is also regulated and these processes are affected by interaction with other
enzymes, including Lon protease, succinyl-CoA synthetase (SCS), and perhaps ferrochelatase (FECH), the
final two also critical enzymes in heme synthesis. Importantly, we recently discovered that ALAS activity is also
dramatically stimulated by mitochondrial ClpX (mtClpX), a member of the AAA+ family of protein unfoldases.
The mtClpX energy-dependent unfoldase accelerates incorporation of PLP into ALAS and CLPX depletion
causes anemia in vertebrates. We also solved structures of both PLP-free ALAS (from yeast) and the active
PLP-bound enzyme, which illuminates the conformational changes coupled to PLP incorporation and provides
important information for understanding mtClpX-promoted loading of PLP. These structures also provide the
first observation of the eukaryotic-specific regulatory C-terminal domain of the enzyme. This domain structure
suggests testable mechanisms to explain the XLPP mutations and contains the binding site for SCS, which we
will further study. Continuing to investigate how mtClpX physically interacts with ALAS and to test models for
the mechanism of PLP-loading holds promise for uncovering a link between mtClpX-ALAS2 interactions and
some classes of XLSA alleles. In another recent, exciting breakthrough, our collaborators discovered a
dominant human CLPX mutation that appears to hyperactivate ALAS, leading to mtClpX-linked erythropoietic
protoporphyria (EPP). The mechanistic basis of this disease will be scrutinized at the molecular, structural and
cellular level. Thus, by probing the complex mechanisms that control ALAS enzymes we will elucidate new
mol...

## Key facts

- **NIH application ID:** 10007817
- **Project number:** 5R01DK115558-04
- **Recipient organization:** MASSACHUSETTS INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** TANIA A BAKER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $418,367
- **Award type:** 5
- **Project period:** 2017-09-15 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10007817

## Citation

> US National Institutes of Health, RePORTER application 10007817, Macromolecular interactions controlling the ALA synthases, keystone enzymes that initiate heme biosynthesis (5R01DK115558-04). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10007817. Licensed CC0.

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