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

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.

Cuprotosis-related signature predicts overall survival in clear cell renal cell carcinoma

“…A study found that FDX1 can impact the prognosis of lung adenocarcinoma (Zhang et al, 2021). LIAS encodes lipoyl synthase, an Fe-S cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily, which is a vital component of the lipoic acid pathway (Hendricks et al, 2021). DLAT encodes dihydrolipoamide S-acetyltransferase (DLAT), which is a subunit of the pyruvate dehydrogenase (PDH) complex and a protein target of lipoylation.…”

Molecular Basis of Rare Diseases Associated to the Maturation of Mitochondrial [4Fe-4S]-Containing Proteins

“…LIAS is a member of the radical S-adenosylmethionine (SAM) superfamily [ 34 , 35 ]. It catalyzes the final step of the biosynthesis of lipoyl cofactor [ 36 , 37 , 38 ] and binds two [4Fe-4S] clusters [ 39 , 40 ]: a [4Fe-4S] cluster (FeS RS ), typical of all radical SAM enzymes, and a [4Fe-4S] cluster (FeS aux ) that provides two sulfur atoms to the lipoyl cofactor [ 41 , 42 ]. It has been shown that the C-domain of NFU1 drives first [4Fe-4S] 2+ cluster delivery from the ISCA1-ISCA2 complex, where the [4Fe-4S] 2+ cluster is assembled, to a [4Fe-4S] 2+ ISCA1-NFU1 intermediate complex, which then specifically directs the cluster into the FeS RS site of LIAS [ 32 , 33 ].…”

Bert Vallee—A 20th Century Adventure(r) in Zincology

“…Bert Vallee witnessed the early days of this metallomics research at the turn of the millennium, although he could no longer actively contribute to it. In his honour, Hendricks et al discuss the role and relevance of thiolate clusters of proteins in different taxonomic groups and report on such a controlled metal insertion procedure [24]. In their manuscript, they show the transfer of iron ions from the iron-sulfur cluster assembly proteins ISCA2 and ISCU to lipoyl synthase (LIAS) [24].…”

“…In his honour, Hendricks et al discuss the role and relevance of thiolate clusters of proteins in different taxonomic groups and report on such a controlled metal insertion procedure [24]. In their manuscript, they show the transfer of iron ions from the iron-sulfur cluster assembly proteins ISCA2 and ISCU to lipoyl synthase (LIAS) [24]. This enzyme is responsible for the biosynthesis of lipoic acid, which has several important biological roles.…”