Biology subjects

Pandelia, M.-E.

Publications and source records attributed to Pandelia, M.-E..

6 recordsLinked to original sources

bioRxiv2026

Amino Acids in the RSSY Motif of Lipoyl Synthase Control Substrate Binding and Reactivity

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 in 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 notable were the S308C and S308A variants, which 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 during 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. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/727706v2_ufig2.gif" ALT="Figure 2"> View larger version (36K): org.highwire.dtl.DTLVardef@19e1f29org.highwire.dtl.DTLVardef@ff1e4org.highwire.dtl.DTLVardef@d1cf91org.highwire.dtl.DTLVardef@923d1d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Transition metal activation reframes SAMHD1 regulation

SAMHD1 is the lone human dNTP triphosphohydrolase and is intimately linked to HIV viral restriction, dNTP pool maintenance, resistance to chemotherapy, and the autoinflammatory Aicardi-Goutieres syndrome. While its substrate promiscuity and nucleotide basis of activity have been extensively studied, the identity and mechanistic roles of its metal cofactors remain poorly defined. Here, we integrate elemental analysis, spectroscopy, protein cross-linking, and enzyme kinetics to elucidate the molecular mechanisms underlying metal-dependent activation and catalysis in SAMHD1. Our findings establish that transition metals are essential components of SAMHD1 function, highlight their overlooked role in allosteric regulation, and reveal a central role for iron in organizing the dinuclear active site. We show that iron is preferentially incorporated in one position of the bimetallic core, where it promotes recruitment of a second divalent metal ion required for activity. While manganese can substitute for iron, it alters the metal binding equilibria, highlighting the unique functional properties of iron. Notably, SAMHD1 exhibits metal cofactor promiscuity at the second metal site, accommodating diverse divalent metals with distinct effects on activity. Cumulatively, our findings establish iron as a core structural and functional determinant of SAMHD1 catalysis and reveal how transition metal selectivity and flexibility enable enzymatic activity across diverse cellular environments and metal flux conditions. Significance StatementSAMHD1 is a central regulator of cellular dNTP poolsand an essential antiviral restriction factor, yet its metal dependence remains poorly defined. Here, we show that SAMHD1 is not a magnesium-driven enzyme but a transition-metal-dependent hydrolase in which iron plays a central structural and regulatory role. We define the metal requirements of the active and allosteric sites and demonstrate that diiron and heterodinuclear iron-containing cofactors form in solution and support catalysis. Transition metals such as iron and manganese act as more effective activators than magnesium, while plasticity at the second metal-binding site enables activity across dynamic metalation and oxidation states. SAMHD1 can thus flexibly tune antiviral defense and nucleotide metabolism, bypassing constraints imposed by metal availability and the cellular redox environment.

biochemistry↗

bioRxiv2026

Promiscuous Metal Site in Hepatitis B Virus X Protein Binds anFe-S Cluster

The Hepatitis B virus (HBV) regulatory protein HBx is essential for viral replication and pathogenesis, yet its cofactor specificity and ligand environment remain poorly defined. Although HBx binds either an Fe-S cluster or Zn, its intrinsic disorder and mutational tolerance have hindered its precise characterization. Here, we integrate chemoproteomics with HYSCORE spectroscopy to identify the metal-coordinating ligands in HBx. Histidine coordination is excluded, while C61, C69, C143, and C148 emerge as primary cysteine ligands for the Fe-S cluster, with C137 acting as a conditional ligand. These residues also bind Zn and are associated with HBx transactivation and clinically relevant variants. HBx engages the host cytosolic Fe-S machinery and displays sensitivity to Fe-S-targeting reagents, behavior consistent with Fe-S cluster acquisition and lability. Together, these findings suggest that HBx functionally behaves as an Fe-S cluster-associated protein, highlighting a potentially druggable vulnerability in HBV replication. Significance StatementFe-S clusters are emerging as key cofactors in viral replication but are often mistaken for Zn due to O2-sensitivity and shared cysteine coordination. The Hepatitis B virus HBx protein, essential for viral replication and hepatocarcinogenesis, has long been mechanistically intractable, with debate over its metallocofactor. Here, we provide evidence that HBx coordinates an Fe-S cluster, placing it within the growing family of viral Fe-S-cluster-containing proteins. Using chemoproteomics, we identify its cysteine ligands, overcoming limitations of mutational analysis in disordered proteins. Although HBx binds both an Fe-S cluster and Zn, its interaction with human Fe-S assembly factors suggests a functional link to Fe-S cluster biology, while its sensitivity to TEMPOL and NO reveals a potentially druggable vulnerabilityin HBx.

biochemistry↗

bioRxiv2026

Discovery of a cluster in the PRRSV Nsp1α leader protease reveals host-virus interplay in its downstream functions

Porcine reproductive and respiratory syndrome virus (PRRSV; Betaarterivirus suid) is a major global threat to swine production, yet effective antiviral therapies are lacking. The leader protease Nsp1 is essential for viral replication and innate immune suppression, and its N-terminal zinc-finger (ZF) domain is critical for function, although its molecular role remains unclear. Here, we show that the ZF domain plays only a minor role in protease activity and that Nsp1 is largely inactive following release from the polyprotein. Using Mossbauer and UV/visible spectroscopy combined with chemoproteomics, we demonstrate that the ZF site binds not only Zn but also a [4Fe-4S] cluster. Notably, the Fe-S cluster, but not Zn, allosterically modulates residual protease activity. Nsp1 directly engages the cytosolic iron-sulfur cluster assembly machinery via CIAO1 and competes with the Fe-S carrier CIAO3, establishing the [4Fe-4S] cluster as a bona fide cofactor. These findings redefine Nsp1 as an Fe-S-dependent viral protein and reveal new opportunities for metal-targeted antiviral strategies.

biochemistry↗

bioRxiv2025

Feedback regulation of iron-sulfur cluster biogenesis

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors in biological systems. Given their central role in bacterial metabolism and pathogenesis, the biogenesis of Fe-S clusters is tightly controlled. We reveal a feedback regulatory mechanism involving the sulfide producing SufS/SufU complex within the sulfur utilization (SUF) system of Mycobacterium tuberculosis, the bacterium that causes tuberculosis. In this mechanism, [2Fe-2S] clusters compete with zinc ions for binding to the sulfide transfer protein SufU. Cluster binding induces SufU tetramerization, which prevents its interaction with the cysteine desulfurase SufS, thereby inhibiting SufS activation and limiting sulfide supply for Fe-S cluster biogenesis. These findings uncover an unrecognized regulatory mechanism in M. tuberculosis, ensuring strict control of Fe-S cluster production.

biochemistry↗

bioRxiv2023

Oxidative rearrangement of tryptophan to indole nitrile by a single diiron enzyme

Nitriles are uncommon in nature and are typically constructed from oximes via the oxidative decarboxylation of amino acid substrates or from the derivatization of carboxylic acids. Here we report a third strategy of nitrile biosynthesis featuring the cyanobacterial nitrile synthase AetD. During the biosynthesis of the eagle-killing neurotoxin, aetokthonotoxin, AetD converts the alanyl side chain of 5,7-dibromo-L-tryptophan to a nitrile. Employing a combination of structural, biochemical, and biophysical techniques, we characterized AetD as a non-heme diiron enzyme that belongs to the emerging Heme Oxygenase-like Diiron Oxidase and Oxygenase (HDO) superfamily. High-resolution crystal structures of AetD together with the identification of catalytically relevant products provide mechanistic insights into how AetD affords this unique transformation that we propose proceeds via an aziridine intermediate. Our work presents a new paradigm for nitrile biogenesis and portrays a substrate binding and metallocofactor assembly mechanism that may be shared among other HDO enzymes.

biochemistry↗