Characterization and Reconstitution of Human Lipoyl Synthase (LIAS) Supports ISCA2 and ISCU as Primary Cluster Donors and an Ordered Mechanism of Cluster Assembly.

James A Cowan,Amber L Hendricks,Christine Wachnowsky,Insiya Fidai,Brian Fries

doi:10.3390/ijms22041598

James A Cowan, Amber L Hendricks + Show 3 more

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https://doi.org/10.3390/ijms22041598

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Abstract

Lipoyl synthase (LIAS) is an iron–sulfur cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. The enzyme contains two [4Fe–4S] centers (reducing and auxiliary clusters) that promote radical formation and sulfur transfer, respectively. Most information concerning LIAS and its mechanism has been determined from prokaryotic enzymes. Herein, we detail the expression, isolation, and characterization of human LIAS, its reactivity, and evaluation of natural iron–sulfur (Fe–S) cluster reconstitution mechanisms. Cluster donation by a number of possible cluster donor proteins and heterodimeric complexes has been evaluated. [2Fe–2S]-cluster-bound forms of human ISCU and ISCA2 were found capable of reconstituting human LIAS, such that complete product turnover was enabled for LIAS, as monitored via a liquid chromatography–mass spectrometry (LC–MS) assay. Electron paramagnetic resonance (EPR) studies of native LIAS and substituted derivatives that lacked the ability to bind one or the other of LIAS’s two [4Fe–4S] clusters revealed a likely order of cluster addition, with the auxiliary cluster preceding the reducing [4Fe–4S] center. These results detail the trafficking of Fe–S clusters in human cells and highlight differences with respect to bacterial LIAS analogs. Likely in vivo Fe–S cluster donors to LIAS are identified, with possible connections to human disease states, and a mechanistic ordering of [4Fe–4S] cluster reconstitution is evident.

Highlights

Lipoic acid is a vital metabolite that is involved in a diverse set of biochemical roles, such as antioxidants and performing a variety of cellular functions in multiple metabolic pathways
The activity of isolated bacterial Lipoyl synthase (LIAS) had historically been limited to one turnover in vitro, but multiple turnovers have been observed for the Escherichia coli (E. coli) enzyme following introduction of iron–sulfur (Fe–S) cluster donor proteins that facilitate reactivation of the auxiliary cluster [7,8]
Based on the results of Electron paramagnetic resonance (EPR) (Figure 1), UV–Vis, and iron quantitation data, it appears that immediately following purification, native recombinant LIAS is purified with one bound [4Fe–4S] cluster, in comparison to bacterial LIAS which purifies with seven equivalents of Fe and six equivalents of S, occupying or partially occupying both sites (Supplementary Figure S1, Supplementary Table S1) [9]

Summary

Introduction

Lipoic acid is a vital metabolite that is involved in a diverse set of biochemical roles, such as antioxidants and performing a variety of cellular functions in multiple metabolic pathways. The activity of isolated bacterial LIAS had historically been limited to one turnover in vitro, but multiple turnovers have been observed for the Escherichia coli (E. coli) enzyme following introduction of iron–sulfur (Fe–S) cluster donor proteins that facilitate reactivation of the auxiliary cluster [7,8]. This reflects the need to refresh the auxiliary cluster, which is the source of sulfur for the insertion reactions. The other (reducing) cluster promotes radical formation on S-adenosylmethionine (SAM) and produces a 5’-deoxyadenosine (5’-dA) radical that abstracts hydrogen from the octanoyl sidechain at the site for subsequent sulfur insertion [9]

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