Amino Acids in the RSSY Motif of Lipoyl Synthase Control Substrate Binding and Reactivity.
6 affiliations
- 1Department of Biochemistry & Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, United States.
- 2Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States.
- 3Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02453, United States.
- 4Department of Chemistry, School of Arts and Sciences, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
- 5Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
- 6Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, United States.
Abstract
The last step in the biosynthesis of the lipoyl cofactor (LipCo) is the addition of two sulfur atoms at C6 and C8 of an n-octanoyl chain attached by an amide linkage to a target lysyl residue of a lipoyl carrier protein. This reaction is catalyzed by lipoyl synthase, a member of the radical S-adenosylmethionine (SAM) superfamily. Lipoyl synthase requires two [4Fe-4S] clusters. One cluster is used to cleave SAM reductively to generate two 5'-deoxyadenosyl 5'-radicals (5'-dA•), which abstract the C6 and C8 hydrogen atoms (H•) of the substrate in two sequential steps. The second cluster, termed the auxiliary cluster, is consumed during turnover to provide the attached sulfur atoms. The auxiliary cluster is ligated by three cysteines in a CX₄CX₅C motif and one serine residue (Ser308 in Escherichia coli) in a highly conserved R³⁰⁶SS³⁰⁸Y motif in the C-terminal region of the protein. Here, we show that Arg306 and Ser308 are absolutely required for LipCo formation. Substitution of Arg306 with Lys results in an essentially inactive protein due to poor substrate binding and positioning in the active site. Multiple substitutions of Ser308 were engineered. Most notably, the S308C and S308A variants greatly diminished LipCo formation. However, the S308C variant resulted in greater production of the 6-mercaptooctanoyl intermediate and the formation of a desaturated product, identified as a 6-octenoyl group. Furthermore, the 3Fe cluster formed during degradation of the auxiliary cluster in the course of C6 sulfur substitution in the wild-type reaction is not observed with the S308C variant. Instead, the auxiliary cluster remains tetranuclear and forms a monothiolated cross-linked species with a high-spin, S = 7/2 configuration that decays to the 6-octenoyl-containing product. Other amino acids in the RSSY motif were not essential for catalysis.
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BibTeX
@article{jeyachandran2026amino,
title = {{Amino Acids in the RSSY Motif of Lipoyl Synthase Control Substrate Binding and Reactivity.}},
author = {Vivian Robert Jeyachandran and Nicholas D Lanz and Maria-Eirini Pandelia and Justin M Rectenwald and Jay V Pendyala and Ravi K Maurya and Amie K Boal and Carsten Krebs and Squire J Booker},
journal = {ACS bio \& med chem Au},
year = {2026},
month = aug,
doi = {10.1021/acsbiomedchemau.6c00105},
url = {https://europepmc.org/article/MED/42921303},
note = {CC BY},
}
RIS
TY - JOUR
TI - Amino Acids in the RSSY Motif of Lipoyl Synthase Control Substrate Binding and Reactivity.
AU - Vivian Robert Jeyachandran
AU - Nicholas D Lanz
AU - Maria-Eirini Pandelia
AU - Justin M Rectenwald
AU - Jay V Pendyala
AU - Ravi K Maurya
AU - Amie K Boal
AU - Carsten Krebs
AU - Squire J Booker
PY - 2026
DA - 2026/08/05
JO - ACS bio & med chem Au
DO - 10.1021/acsbiomedchemau.6c00105
UR - https://europepmc.org/article/MED/42921303
AB - The last step in the biosynthesis of the lipoyl cofactor (LipCo) is the addition of two sulfur atoms at C6 and C8 of an n-octanoyl chain attached by an amide linkage to a target lysyl residue of a lipoyl carrier protein. This reaction is catalyzed by lipoyl synthase, a member of the radical S-adenosylmethionine (SAM) superfamily. Lipoyl synthase requires two [4Fe-4S] clusters. One cluster is used to cleave SAM reductively to generate two 5'-deoxyadenosyl 5'-radicals (5'-dA•), which abstract the C6 and C8 hydrogen atoms (H•) of the substrate in two sequential steps. The second cluster, termed the auxiliary cluster, is consumed during turnover to provide the attached sulfur atoms. The auxiliary cluster is ligated by three cysteines in a CX₄CX₅C motif and one serine residue (Ser308 in Escherichia coli) in a highly conserved R³⁰⁶SS³⁰⁸Y motif in the C-terminal region of the protein. Here, we show that Arg306 and Ser308 are absolutely required for LipCo formation. Substitution of Arg306 with Lys results in an essentially inactive protein due to poor substrate binding and positioning in the active site. Multiple substitutions of Ser308 were engineered. Most notably, the S308C and S308A variants greatly diminished LipCo formation. However, the S308C variant resulted in greater production of the 6-mercaptooctanoyl intermediate and the formation of a desaturated product, identified as a 6-octenoyl group. Furthermore, the 3Fe cluster formed during degradation of the auxiliary cluster in the course of C6 sulfur substitution in the wild-type reaction is not observed with the S308C variant. Instead, the auxiliary cluster remains tetranuclear and forms a monothiolated cross-linked species with a high-spin, S = 7/2 configuration that decays to the 6-octenoyl-containing product. Other amino acids in the RSSY motif were not essential for catalysis.
SN - 2694-2437
ER -