Biology subjects

Serebryakova, M. V.

Publications and source records attributed to Serebryakova, M. V..

7 recordsLinked to original sources

bioRxiv2026

Na+-translocating oxaloacetate decarboxylase from Vibrio cholerae: the functional tautomeric form of the substrate and the proton pathways in catalysis

Membrane-bound decarboxylases couple carboxylic acid decarboxylation to the transport of Na+ ions out of prokaryotic cells. The molecular mechanism of decarboxylase action is not yet known, which contrasts with the progress achieved in studying other primary ion pumps. Measuring decarboxylase activity is complicated by slow keto-enol tautomerization of the substrates during the assay. We found that HEPES exhibits anomalously high efficiency as a general acid catalyst for C-H bond formation during the enol-to-ketone conversion of oxaloacetate. Accordingly, the addition of HEPES to the assay medium eliminated the contribution of tautomerization rate to measured decarboxylation rate. Using the dependence of oxaloacetate tautomerization rate and equilibrium on solvent properties and pH, we established that only the keto form of oxaloacetate is converted by Vibrio cholerae oxaloacetate decarboxylase. Steady-state kinetic measurements did not reveal cooperativity in oxaloacetate conversion and Na+ binding. The effects of ionophores (CCCP, valinomycin, and ETH157) on proton transport in pyranine-loaded membrane vesicles prepared from V. cholerae cells indicated that the proton required for the conversion of oxaloacetate to pyruvate is taken up from the cytoplasmic side of the membrane. Furthermore, the effects suggested that {Delta}pH generation is caused by secondary electrophoretic proton transport in exchange for Na+.These findings advance our understanding of the molecular mechanism of the decarboxylation-supported Na+ transport in bacteria.

biochemistry↗

bioRxiv2026

Trimethylammonia-lyases of Shewanella oneidensis and Their Role in Anaerobic Respiration

The facultatively anaerobic bacterium Shewanella oneidensis MR-1 contains in its genome two operons, so_3056-3058 and so_3299-3301, each including genes for putative periplasmic flavocytochrome c and ammonia-lyase of aromatic amino acids. To determine their role in anaerobic respiration, we produced the encoded ammonia-lyases SO_3057 and SO_3299 in Escherichia coli and determined their substrate specificities. SO_3057 was found to cleave trimethylammonium group from ergothioneine to yield thiourocanic acid, whereas SO_3299 catalyzed a similar conversion of N({pi})-methyl histidine betaine to yield N({pi})-methyl urocanate. The catalytic efficiencies (kcat/Km values) were (3-4) x 106 M-1 s-1, and the pH optima of activity were between 8 and 9. Ergothioneine induced SO_3057 synthesis in anaerobic S. oneidensis cells and their growth, and thiourocanate stimulated respiration as an alternative terminal electron acceptor. The predicted 3D structures of the genetically coupled flavocytochromes c (SO_3056/58 and SO_3300/3301) are consistent with their use of thiourocanate and N({pi})-methyl urocanate, respectively, as electron acceptors. We therefore conclude that the periplasmic lyases encoded by the so_3057 and so_3299 genes contribute to anaerobic respiration in S. oneidensis by producing terminal electron acceptors for the genetically coupled flavocytochromes c.

microbiology↗

bioRxiv2026

A Unique Type V CRISPR-Cas System Encoded by a Group of Thermus Viruses

CRISPR-Cas are widespread adaptive immune systems that protect bacteria and archaea from mobile genetic elements such as bacteriophages. Metagenomic sequencing identified CRISPR-Cas systems in phage genomes; however, their functions remain largely unknown. Here, we present Cas12r-CRISPR, a novel type V CRISPR-Cas system encoded by Lalka phages infecting thermophilic Thermus bacteria. We determined Cas12r-CRISPR PAM consensus sequence and crRNA structure and showed, that when provided with appropriate spacers and expressed in Thermus thermophilus, Cas12r-CRISPR efficiently interferes with plasmid transformation as well as infection by diverse Thermus phages. In the course of Lalka phage infection, the Cas12r-CRISPR locus is expressed with middle phage genes and its transcripts are among the most abundant phage RNAs. Notably, most Cas12r-CRISPR spacers target integrative mobile elements widespread in Thermus genomes. Both Lalka phages and targeted integrative mobile elements use host tRNA genes as attachment sites. We therefore propose that Cas12r-CRISPR participates in an inter-MGEs conflict. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/719267v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@f90c35org.highwire.dtl.DTLVardef@90a063org.highwire.dtl.DTLVardef@111f936org.highwire.dtl.DTLVardef@1008132_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Histidine betaine trimethylammonia-lyase, a novel enzyme coupled with terminal urocanate reductase in Shewanella woodyi grown anaerobically

Bacteria coping with oxygen deficiency can switch to alternative terminal electron acceptors, which can be normal metabolic intermediates or products of dedicated coupled reactions. In the latter case, the genes for the respective terminal reductase and coupling enzyme are expected to cluster in the genome. Here, we determined the roles of two uncharacterized periplasmic proteins encoded by the swoo_3912-swoo_3913 gene cluster in the facultatively anaerobic marine bacterium Shewanella woodyi. We confirmed the current database annotation of the former protein as "urocanate reductase" but identified the latter protein as a previously unknown histidine betaine trimethylammonia-lyase (HBTL) enzyme. HBTL converts histidine betaine into urocanate and trimethylamine and is remarkably specific for histidine betaine as substrate. HBTL requires Mg2+ for activity and undergoes slow reversible inactivation at low Mg2+ concentrations. HBTL activity was not evident in S. woodyi cells grown aerobically but was induced in cells grown anaerobically. Both histidine betaine and urocanate supported anaerobic S. woodyi growth and, hence, respiration. Similar gene clusters are found in many anaerobic bacteria, suggesting a wide occurrence of the anaerobic respiration pathway discovered in this work in the bacterial world.

biochemistry↗

bioRxiv2024

Rapid kinetics of H+ transport by membrane pyrophosphatase: evidence for a direct-coupling mechanism

Stress resistance-conferring membrane pyrophosphatase (mPPase) found in microbes and plants couples pyrophosphate hydrolysis with H+ transport out of the cytoplasm. There are two opposing views on the energy-coupling mechanism in this transporter: the pumping is associated with either pyrophosphate binding to mPPase or the hydrolysis step. We used our recently developed stopped-flow pyranine assay to measure H+ transport into mPPase-containing inverted membrane vesicles on the timescale of a single turnover. The vesicles were prepared from Escherichia coli overproducing the H+-translocating mPPase of Desulfitobacterium hafniense. Pyrophosphate induced linear accumulation of H+ in the vesicles, without evident lag or burst. In contrast, the binding of three nonhydrolyzable pyrophosphate analogs essentially induced no H+ accumulation. These findings are inconsistent with the "pumping-before-hydrolysis" model of mPPase functioning and support the alternative model positing the hydrolysis reaction as the source of the transported H+ ions. mPPase is thus a first "directly-coupled" proton pump.

biochemistry↗

bioRxiv2024

Acrylate reductase of an anaerobic electron transport chain of the marine bacterium Shewanella woodyi

Many microorganisms are capable of anaerobic respiration in the absence of oxygen, by using different organic compounds as terminal acceptors in electron transport chain. We identify here an anaerobic respiratory chain protein responsible for acrylate reduction in the marine bacterium Shewanella woodyi. When the periplasmic proteins of S. woodyi were separated by ion exchange chromatography, acrylate reductase activity copurified with an ArdA protein (Swoo_0275). Heterologous expression of S. woodyi ardA gene (swoo_0275) in Shewanella oneidensis MR-1 cells did not result in the appearance in them of periplasmic acrylate reductase activity, but such activity was detected when the ardA gene was co-expressed with an ardB gene (swoo_0276). Together, these genes encode flavocytochrome c ArdAB, which is thus responsible for acrylate reduction in S. woodyi cells. ArdAB was highly specific for acrylate as substrate and reduced only methacrylate (at a 22-fold lower rate) among a series of other tested 2-enoates. In line with these findings, acrylate and methacrylate induced ardA gene expression in S. woodyi under anaerobic conditions, which was accompanied by the appearance of periplasmic acrylate reductase activity. ArdAB-linked acrylate reduction supports dimethylsulfoniopropionate-dependent anaerobic respiration in S. woodyi and, possibly, other marine bacteria.

biochemistry↗

bioRxiv2020

Unprecedentedly efficient CUG initiation of an overlapping reading frame in POLG mRNA yields novel protein POLGARF

While near cognate codons are frequently used for translation initiation in eukaryotes, their efficiencies are usually low (<10% compared to an AUG in optimal context). Here we describe a rare case of highly efficient near cognate initiation. A CUG triplet located in the 5 leader of POLG mRNA initiates almost as efficiently (~60-70%) as an AUG in optimal context. This CUG directs translation of a conserved 260 triplet-long overlapping ORF, which we call POLGARF (POLG Alternative Reading Frame). Translation of a short upstream ORF 5 of this CUG governs the ratio between DNA polymerase and POLGARF produced from a single POLG mRNA. Functional investigation of POLGARF points to extracellular signalling. While unprocessed POLGARF resides in the nucleoli together with its interacting partner C1QBP, serum stimulation results in rapid secretion of POLGARF C-terminal fragment. Phylogenetic analysis shows that POLGARF evolved ~160 million years ago due to an MIR transposition into the 5 leader sequence of the mammalian POLG gene which became fixed in placental mammals. The discovery of POLGARF unveils a previously undescribed mechanism of de novo protein-coding gene evolution. Significance StatementIn this study, we describe previously unknown mechanism of de novo protein-coding gene evolution. We show that the POLG gene, which encodes the catalytic subunit of mitochondrial DNA polymerase, is in fact a dual coding gene. Ribosome profiling, phylogenetic conservation, and reporter construct analyses all demonstrate that POLG mRNA possesses a conserved CUG codon which serves as a start of translation for an exceptionally long overlapping open reading frame (260 codons in human) present in all placental mammals. We called the protein encoded in this alternative reading frame POLGARF. We provide evidence that the evolution of POLGARF was incepted upon insertion of an MIR transposable element of the SINE family.

molecular biology↗