Biology subjects

Silakov, A.

Publications and source records attributed to Silakov, A..

6 recordsLinked to original sources

bioRxiv2026

Metal Coordination Dynamics Governs Selective Epoxidation in Hyoscyamine 6β-Hydroxylase: Integrated Experimental and Computational Insights

Iron(II)- and 2-oxoglutarate-dependent (Fe(II)/2OG) enzymes catalyze a wide range of C-H bond activation and functionalization reactions and play essential roles in biosynthesis and metabolic regulation. Despite extensive mechanistic studies, the principles governing selectivity between canonical hydroxylation and alternative transformations remain incompletely understood. Here, we investigate the catalytic mechanism of hyoscyamine 6{beta}-hydroxylase (H6H), a Fe(II)/2OG-dependent oxygenase that sequentially catalyzes the 6{beta}-hydroxylation of hyoscyamine followed by 6,7-exo-epoxidation of 6{beta}-hydroxyhyoscyamine to generate scopolamine. Combined molecular dynamics and QM/MM calculations reveal that an inline Fe(IV)-oxo intermediate initiates hydrogen atom abstraction from the substrate C7 position. The resulting Fe(III)-OH species subsequently deprotonates the substrate hydroxyl group in a process coupled to substrate coordination to the iron center and an in-line-to-off-line rearrangement of the Fe(III)-OH moiety. This coordination dynamics machinery is further supported by the observed chlorination reactivity on the same substrate. Importantly, this coordination switch favors epoxide formation over hydroxyl rebound, thereby directing the reaction toward selective epoxidation. Further computational analysis of the L290F variant demonstrates that steric constraints imposed by L290 are essential for suppressing hydroxylation, revealing a bidirectional regulatory mechanism governing epoxidation/hydroxylation selectivity. Whereas iron coordination dynamics promote epoxidation reactivity, precise substrate positioning and protein-derived steric effects suppress the competing hydroxylation pathway. These findings are consistent with available experimental observations and establish metal coordination dynamics as a key determinant of selective C-H functionalization in Fe(II)/2OG enzymes.

biochemistry↗

bioRxiv2026

Redox Regulation in O2-Tolerant Hydrogenases: Insights from two homologues.

O2-tolerance is a desirable property for [FeFe] hydrogenases, which are highly efficient H2-producing catalysts. While most such enzymes are highly sensitive to aerobic environments, a small number of explored representatives exhibit exceptional stability and even H2-producing activity under oxygenic conditions. However, the genetic signatures of the O2-tolerance in this class of enzymes remain largely unknown. To address this knowledge gap, we explored a close homologue of a well-characterized O2-tolerant [FeFe] hydrogenase from Clostridium beijerinckii (CbHydA1) - a hydrogenase from Terrisporobacter glycolicus (TgHydA1). Our investigation indeed confirms that TgHydA1 can transition to the O2-stable Hinact state, a hallmark of O2 tolerance. The surprising outcome is that despite the high amino acid similarity, TgHydA1 shows a substantially higher propensity to remain in the Hinact state than CbHydA1. Using protein film electrochemical experiments, we demonstrate that the root of this behavior lies in roughly tenfold slower reactivation rates than those of CbHydA1 at any applied potential. This degree and direction of variation in reactivation kinetics have not been observed before for any other O2-tolerant [FeFe] hydrogenases or their variants to date, uncovering a yet-to-be-explored facet of reactivity alteration available to these enzymes. Overall, the results presented here highlight the importance of a holistic analysis of [FeFe] hydrogenase sequences in the context of their interaction with O2 that encompasses the protein environment and properties of the auxiliary metallocofactors.

biochemistry↗

bioRxiv2026

Evidence for an Organometallic Species Formed in the ArsL Reaction

The radical S-adenosylmethionine (SAM) superfamily comprises over 800,000 unique sequences of enzymes that catalyze more than 100 distinct reactions. Canonical radical SAM (RS) enzymes are composed of a full or partial triose phosphate isomerase fold and contain a highly conserved CX3CX2C motif. The cysteines in the conserved motif ligate an [Fe4S4] cluster used in the reductive cleavage of SAM to yield methionine and a 5'-deoxyadenosyl 5'-radical (5'-dA*). The 5'-dA* is typically used to initiate catalysis by abstracting a hydrogen atom from a bound substrate, yielding 5'-deoxyadenosine and a substrate radical. ArsL, a recently characterized RS enzyme involved in the biosynthesis of the antibiotic arsinothricin (AST), catalyzes a reaction that deviates from the canonical RS reaction, forming methylthioadenosine and a 3-amino-3-carboxypropyl radical (ACP*). The ACP* is used to construct a carbon-arsenic bond to form the organoarsenic compound hydroxyarsinothricin (AST-OH), the penultimate enzymatic step in forming AST. While investigating how ArsL suppresses the formation of the 5'-dA* in favor of the ACP*, we discovered that ArsL forms a unique organometallic species containing a bond between the gamma carbon of ACP and the unique iron of the [Fe4S4] cluster. We propose that ArsL uses this novel species to generate a sufficiently electrophilic carbon that can be attacked by arsenous acid, thereby forming the carbon-arsenic bond.

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

Structural and Spectroscopic Basis for Catalysis by a Class C Radical S-adenosylmethionine Methylase Involved in Nosiheptide/Nocathiacin Biosynthesis

Nosiheptide (NOS) is a ribosomally synthesized and post-translationally modified peptide (RiPP) natural product that exhibits potent antibiotic activity against multiple bacterial pathogens. NOS features a core macrocyclic peptide containing thiazoles, dehydrated serine and threonine residues, and a 3-hydroxypyridine ring. In addition to the macrocycle, NOS possesses a side-ring system formed by a 3-methyl-2-indolic acid (MIA) bridge that connects to glutamyl and cysteinyl residues on the core peptide via ester and thioester linkages. This unique side-ring is installed by the class C radical S-adenosylmethionine (SAM) methylase NosN. Here, we report three X-ray crystal structures of the NosN homolog, NocN, at resolutions of 1.4 [A], 1.84 [A], and 1.78 [A] under anaerobic conditions, representing the first structural characterization of a class C radical SAM methylase. The structures reveal clear electron density for two bound SAM molecules. Remarkably, the C5' atom of SAMI, which coordinates to the [Fe4S4] cluster, lies 3.5 [A] from the methyl group of SAMII and is properly positioned for direct hydrogen atom abstraction. A structure containing a product mimic illustrates how NocN engages its substrate and identifies Tyr276 as a key catalytic residue. The structure further suggests that the sulfonium center of SAMII may undergo epimerization to facilitate radical attack. Finally, electron paramagnetic resonance spectroscopy identifies a paramagnetic species consistent with the addition of the SAMII-derived methylene radical to the MIA substrate.

biochemistry↗

bioRxiv2023

Synergistic Binding of the Halide and Cationic Prime Substrate of the L-Lysine 4-Chlorinase, BesD, in Both Ferrous and Ferryl States

An aliphatic halogenase requires four substrates: 2-oxoglutarate (2OG), halide (Cl- or Br-), the halogenation target ("prime substrate"), and dioxygen. In well-studied cases, the three non-gaseous substrates must bind to activate the enzymes Fe(II) cofactor for efficient capture of O2. Halide, 2OG, and (lastly) O2 all coordinate directly to the cofactor to initiate its conversion to a cis-halo-oxo-iron(IV) (haloferryl) complex, which abstracts hydrogen (H*) from the non-coordinating prime substrate to enable radicaloid carbon-halogen coupling. We dissected the kinetic pathway and thermodynamic linkage in binding of the first three substrates of the O_SCPLOWLC_SCPLOW-lysine 4-chlorinase, BesD. After 2OG adds, subsequent coordination of the halide to the cofactor and binding of cationic O_SCPLOWLC_SCPLOW-Lys near the cofactor are associated with strong heterotropic cooperativity. Progression to the haloferryl intermediate upon addition of O2 does not trap the substrates in the active site and, in fact, markedly diminishes cooperativity between halide and O_SCPLOWLC_SCPLOW-Lys. The surprising lability of the BesD*[Fe(IV)=O]*Cl*succinate*O_SCPLOWLC_SCPLOW-Lys complex engenders pathways for decay of the haloferryl intermediate that do not result in O_SCPLOWLC_SCPLOW-Lys chlorination, especially at low chloride concentrations; one identified pathway involves oxidation of glycerol. The mechanistic data imply that (i) BesD may have evolved from a hydroxylase ancestor either relatively recently or under weak selective pressure for efficient chlorination and (ii) that acquisition of its activity may have involved the emergence of linkage between O_SCPLOWLC_SCPLOW-Lys binding and chloride coordination following loss of the anionic protein-carboxylate iron ligand present in extant hydroxylases.

biochemistry↗