Biology subjects

Ryzhykau, Y.

Publications and source records attributed to Ryzhykau, Y..

5 recordsLinked to original sources

bioRxiv2025

Topological adaptation of heliorhodopsins enables exogenous second-antenna acquisition in monoderm phototrophs

Heliorhodopsins (HeRs), the third rhodopsin family, are characterized by inverted membrane topology and confinement to monoderm organisms, yet their biological meaning has so far remained a mystery. We report the first crystal structure of a eukaryotic HeR, supported by structural modeling and comparative analyses across all domains of life. A conserved carotenoid-binding site, reminiscent of secondary antennae in some microbial rhodopsins, is identified and found to be common among HeRs. We show that inverted topology allows recruitment of exogenous xanthophylls, inaccessible in diderm cells, explaining HeRs distinctive orientation and distribution. These findings reveal a previously unrecognized light-harvesting mechanism of HeRs, expand the known repertoire of microbial phototrophy, and suggest evolutionary constraints linking membrane topology to environmental metabolite accessibility.

biochemistry↗

bioRxiv2025

Structural and Functional Characterization of Encapsulin-Targeted Double Ferritin Fold Ferroxidases

Ferritins are a widespread family of proteins involved in iron homeostasis. While classic ferritins consist of four -helices and form 24-meric nanocages, related ferritin-like proteins display other types of assemblies and sometimes lack any iron storage capacity. Here, by analyzing the available genomic data, we identify a family of double ferritin-like proteins (DFLPs) composed of two four-helical domains, which arose by duplication of a ferritin fold protein. We characterize representative DFLPs from Thermocrinis minervae and Caldanaerovirga acetigignens, TmDFLP and CaDFLP, and show that they form homodimers and bind heme. We determine the X-ray structure of TmDFLP and demonstrate its ferroxidase activity. Furthermore, we show that some DFLPs, including TmDFLP and CaDFLP, are highly likely to be targeted into encapsulin shells. Our work expands the range of known iron metabolism systems and highlights the power of genome mining for discovery of new proteins. Graphical Abstract for Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/676036v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@72e6b0org.highwire.dtl.DTLVardef@18752fforg.highwire.dtl.DTLVardef@15a02e7org.highwire.dtl.DTLVardef@74cd02_HPS_FORMAT_FIGEXP M_FIG C_FIG A family of double ferritin-like proteins (DFLPs) composed of two four-helical domains is described and investigated. DFLPs are shown to form homodimers, bind heme, possess diiron sites and display ferroxidase activity. Some DFLPs are shown to be targeted to encapsulin shells as core or secondary cargo, thus representing a new type of iron metabolism systems.

biochemistry↗

bioRxiv2024

High-throughput algorithm predicts F-Type ATP synthase rotor ring stoichiometries of 8 to 27 protomers

ATP synthases are large enzymes present in every living cell. They consist of a transmembrane and a soluble domain, each comprising multiple subunits. The transmembrane part contains an oligomeric rotor ring (c-ring), whose stoichiometry defines the ratio between the number of synthesized ATP molecules and the number of ions transported through the membrane. Currently, c-rings of F-Type ATP synthases consisting of 8 to 17 (except 16) subunits have been experimentally demonstrated. Here, we present an easy-to-use high-throughput computational approach based on AlphaFold that allows us to estimate the stoichiometry of all homooligomeric c-rings, whose sequences are present in genomic databases. We validate the approach on the available experimental data, obtaining the correlation as high as 0.94 for the reference data set, and use it to predict the existence of c-rings with stoichiometry varying from 8 to 27. We then conduct molecular dynamics simulations of two c-rings with stoichiometry above 17 to corroborate the machine learning-based predictions. Our work strongly suggests existence of rotor rings with previously undescribed high stoichiometry in natural organisms and highlights the utility of AlphaFold-based approaches for studying homooligomeric proteins.

bioinformatics↗

bioRxiv2023

luxA gene from Enhygromyxa salina encodes a functional homodimeric luciferase

Several clades of luminescent bacteria are known currently. They all contain similar lux operons, which include the genes luxA and luxB encoding a heterodimeric luciferase. The aldehyde oxygenation reaction is catalyzed by the subunit LuxA, while LuxB is inactive. Recently, genomic analysis identified a subset of bacterial species with rearranged lux operons lacking luxB. Here, we show that the product of the luxA gene from the reduced luxACDE operon of Enhygromyxa salina is luminescent upon addition of aldehydes both in vivo in Escherichia coli and in vitro. Overall, EsLuxA is less bright compared with luciferases from Aliivibrio fischeri (AfLuxAB) and Photorhabdus luminescens (PlLuxAB), and most active with medium-chain C4-C9 aldehydes. Crystal structure of EsLuxA determined at the resolution of 2.71 [A] reveals a classical monooxygenase fold, and the protein preferentially forms a dimer in solution. The mobile loop residues 264-293, which form a {beta}-hairpin or a coil in Vibrio harveyi LuxA, form -helices in EsLuxA. Phylogenetic analysis shows EsLuxA and related proteins may be bacterial protoluciferases that arose prior to duplication of the luxA gene and its speciation to luxA and luxB in the previously described luminescent bacteria. Our work paves the way for discovery of new luciferases that have an advantage of being encoded by a single gene.

biochemistry↗

bioRxiv2022

Structural insights into 3Fe-4S ferredoxins diversity in M.tuberculosis highlighted by a first redox complex with P450.

Ferredoxins are small iron-sulfur proteins and key players in essential metabolic pathways. Among all types, 3Fe-4S ferredoxins are less studied mostly due to anaerobic requirements. Their complexes with cytochrome P450 redox partners have not been structurally characterized. In the present work, we solved the structures of both 3Fe-4S ferredoxins from M. tuberculosis - Fdx alone and the fusion FdxE-CYP143. Our SPR analysis demonstrated a high affinity binding of FdxE to CYP143. According to SAXS data, the same complex is present in solution. The structure reveals extended multipoint interactions and the shape/charge complementarity of redox partners. Furthermore, FdxE binding induced conformational changes in CYP143 as evident from the solved CYP143 structure alone. The comparison of FdxE-CYP143 and modeled Fdx-CYP51 complexes further revealed the specificity of ferredoxins. Our results illuminate the diversity of electron transfer complexes for the production of different secondary metabolites.

biochemistry↗