Human aminolevulinate synthase structure reveals a eukaryotic-specific autoinhibitory loop regulating substrate binding and product release

Henry J Bailey,Wyatt W Yue,Robert J Desnick,Siladitya Padhi,Arijit Roy,Gustavo A Bezerra,William R Foster,Elzbieta Rembeza,Harry A Dailey,David F Bishop,Jason R Marcero,Gopalakrishnan Bulusu

doi:10.1038/s41467-020-16586-x

Abstract

5′-aminolevulinate synthase (ALAS) catalyzes the first step in heme biosynthesis, generating 5′-aminolevulinate from glycine and succinyl-CoA. Inherited frameshift indel mutations of human erythroid-specific isozyme ALAS2, within a C-terminal (Ct) extension of its catalytic core that is only present in higher eukaryotes, lead to gain-of-function X-linked protoporphyria (XLP). Here, we report the human ALAS2 crystal structure, revealing that its Ct-extension folds onto the catalytic core, sits atop the active site, and precludes binding of substrate succinyl-CoA. The Ct-extension is therefore an autoinhibitory element that must re-orient during catalysis, as supported by molecular dynamics simulations. Our data explain how Ct deletions in XLP alleviate autoinhibition and increase enzyme activity. Crystallography-based fragment screening reveals a binding hotspot around the Ct-extension, where fragments interfere with the Ct conformational dynamics and inhibit ALAS2 activity. These fragments represent a starting point to develop ALAS2 inhibitors as substrate reduction therapy for porphyria disorders that accumulate toxic heme intermediates.

Highlights

5′-aminolevulinate synthase (ALAS) catalyzes the first step in heme biosynthesis, generating 5′-aminolevulinate from glycine and succinyl-CoA
Frameshift indel mutations in exon 11 of the human ALAS2 gene that result in deletion, replacement, or elongation of its Ctextension are the molecular cause of X-linked protoporphyria (XLP, MIM 300752), an inherited disorder that presents with painful phototoxicity and an increased risk for liver dysfunction and failure[21,22], due to high levels of the toxic heme intermediate protoporphyrin IX (PPIX)
Insect cell-expressed hsALAS2 is active with Km values (31 ± 4 μM for succinyl-CoA, 11.8 ± 1.0 μM for glycine) close to those reported by others using a similar assay[24] (Supplementary Fig. 4a, b)

Summary

Introduction

5′-aminolevulinate synthase (ALAS) catalyzes the first step in heme biosynthesis, generating 5′-aminolevulinate from glycine and succinyl-CoA. The structure of R. capsulatus ALAS (rcALAS) was the first reported for the enzyme[12], revealing an induced-fit mechanism upon substrate binding, via an opento-close transition of a mobile active site loop Conformational mobility of this loop has been proposed to be a kinetic barrier for product release[8,11,13]. Frameshift indel mutations in exon 11 of the human ALAS2 gene (on chromosome Xp11.21) that result in deletion, replacement, or elongation of its Ctextension are the molecular cause of X-linked protoporphyria (XLP, MIM 300752), an inherited disorder that presents with painful phototoxicity and an increased risk for liver dysfunction and failure[21,22], due to high levels of the toxic heme intermediate PPIX. XLSA is attributable to mutations within exons 5–11 (including a few in the Ct-extension; from Human Gene Mutation Database)[32] that are predominantly missense in nature

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Conclusion