Genetically encoded green calcium indicators (GECIs) are broadly used for visualizing calcium transients in living cells. Among the diverse family of green GECIs, the Troponin C-based family offers potential advantages, including reduced calcium buffering, smaller molecular size, linear calcium response, and low cytotoxicity. However, the Troponin C-based GECIs with inverted calcium response are less developed compared to other popular GECIs, including GCaMPs and GECOs, and, as a consequence, have several drawbacks related to low dynamic range, brightness, photostability, and calcium ion sensitivity. To address these limitations, we developed a novel GECI, called icBTnC2, which incorporates Troponin C as a sensing moiety and the new bright photostable green FP mBaoJin as a reporting moiety. icBTnC2 demonstrated an inverted fluorescent response to calcium ion binding with a Kd of 62 nM. In terms of fluorescence contrast and calcium ion affinity in vitro, icBTnC2 was comparable to the best widely used calmodulin-based GECIs from the GCaMP family. icBTnC2 demonstrated superior photostability under wide-field fluorescence microscopy exhibiting 5.5-, 4.8-, 3.2-, 2.9-, and 1.3-fold higher photobleaching half-time compared to iYTnC2, mEGFP, NCaMP7, jGCaMP8f, and mBaoJin, respectively. The icBTnC2 indicator was benchmarked against other GECIs, such as jGCaMP8f, NCaMP7, iYTnC2, and R-GECO1, for visualization of calcium transients in mammalian cells and primary neuron cultures, and tested for calcium-dependent changes in fluorescence lifetime. Finally, we solved the crystal structure of the icBTnC2 indicator at 1.55 Å resolution in the calcium-bound state and, using directed mutagenesis, proposed the molecular basis of its fluorescent response to calcium ion binding.

Influenza remains one of the most common and contagious respiratory infections causing around a billion cases of seasonal illness annually and five million cases of severe consequences. A recently reported G2.3 antibody exhibited a potent cross-subtype activity against Group 1 influenza A viruses. Here, we shed light on the structural basis of the broad neutralizing activity of G2.3 by using cryoEM. Structural analysis of the G2.3 complex with H1 hemagglutinin revealed a partly conserved epitope located on the stem domain. The structural data were confirmed by the assessment of binding and neutralizing properties of the Fc-modified form of G2.3 with a broad panel of recombinant hemagglutinins and influenza A viruses. We demonstrated remarkably high activity of G2.3-Fc against H1N1 viral strains, which is consistent with G2.3 epitope being the most conserved within the H1 subtype, but low activity towards Group 2 of HA, which was explained by the analysis of the epitope. To suggest the mechanism of G2.3 neutralization, we compared its epitope with those of other broadly neutralizing antibodies that attack the stem domain. Finally, we obtained an escape mutant that has two mutations within the epitope and in its vicinity, unseen in circulating H1 strains, allowing this mutant to elude from G2.3 and made assumption of the mechanism of such evasion.

The recently discovered family of microalgal water-soluble astaxanthin-binding proteins (AstaPs) functions in carotenoid sequestration and transfer. We present the crystal structure and characterization of AstaP-pink1, a homolog from Scenedesmus sp. We show that despite low sequence identity with the known AstaP-orange1, AstaP-pink1 also binds various xanthophylls, but in contrast to AstaP-orange1, induces a pronounced bathochromic shift and a near-UV spectral bump upon binding ketocarotenoids, which is reminiscent of photoactivated Orange Carotenoid Protein (OCP). Mutagenesis and domain-swap experiments indicate that its unique spectral tuning likely results from carotenoid isomerization, forced by the interplay between its N-terminal jaw and body subdomains connected by the hinge loop of different sequence and length. This study attempts to rationalize the spectral and functional diversification of AstaPs and highlights their potential as engineered modules for carotenoid delivery.

Thiocyanate dehydrogenase (TcDH) plays a pivotal role in the decomposition of thiocyanate in some sulfur-oxidizing bacteria. Here we report the assembly mechanism of the unique three-copper center (Cu1, Cu2 and Cu3) of TcDH in vitro. Using EPR, we established the sequence of copper incorporation into the active center: copper ions are first incorporated in parallel into Cu1 and Cu2 sites, and finally into Cu3 site. TcDH shows high affinities for both Cu(II) (KD values for three sites range from 2.0 × 10-12 to 3.8 × 10-17 M) and Cu(I) (average KD values of three sites are 1.0 × 10-13 M). The process of assembling the TcDH copper center, and thus acquiring the enzyme catalytic activity, is kinetically fast with Cu(I) (~min) ions but extremely slow with Cu(II) (~hours), which correlates with the rate of Cu(II) incorporation into the protein. The inactivation of TcDH due to removal of copper from the active site is kinetically slow due to the low accessibility of the copper center. This phenomenon appears to act as an adaptation mechanism that prevents unwanted loss of copper in vivo due to interaction with the higher-affinity copper binders. Based on the slow incorporation of Cu(II) into the TcDH active center in vitro, we suggest that it is Cu(I) that is involved in the process of assembling of TcDH copper center in vivo.

In situ enzymatic production of bioactive compounds from their non-toxic precursors is a relatively new strategy in drug design. If used correctly, the enzyme-prodrug binary system allows to obtain the necessary therapeutic concentrations of drugs locally and under controlled release. In nature alliinase uses S-substituted L-cysteine sulfoxide as the key part of the defense mechanisms in plants of the Allium genus, and the resulting biologically active thiosulfinates have various health benefits. In our study we describe the recombinant enzyme cystathionine beta-lyase from Klebsiella pneumoniae (kCBL) and its capability to catalyze the similar process, transforming a wide range of S-alk(en)yl- and arylalkyl-derivatives of L-cysteine sulfoxide into the corresponding thiosulfinates. The reaction proceeds with high catalytic efficiency and reaches up to 100 % conversion of the substrate. Besides a comprehensive biochemical analysis, the 3D structure of the holoenzyme and its complex with S-methyl-L-cysteine sulfoxide have been obtained at 1.9 and 2.0 & Aring; resolution, accordingly. The binding pocket of the enzyme provides suitable environment to catalyze beta-elimination reaction of the substituted Lcysteine sulfoxides of different size. To our knowledge, kCBL is the only described bacterial enzyme with such a wide substrate specificity, which makes it promising to be used as a component of the enzyme-prodrug binary system.

Pyridoxal-5′-phosphate (PLP)-dependent D-amino acid transaminases (DATAs) catalyze stereoselective transfer of an amino group from a D-amino acid to an α-keto acid to form new D-amino acid and α-keto acid. These enzymes are found in bacteria and plants; they are responsible for the synthesis of D-amino acids and are incorporated into the nitrogen cycle. In general, the mechanism of D-transamination is similar to the known mechanism of transamination for aspartate aminotransferase: D-transamination reaction consists of two half-reactions with intermediate transfer of the amino group to the cofactor and formation of its reduced form, pyridoxamine-5′-phosphate. DATAs are characterized by broad substrate specificity and an open active site, which, however, does not affect their high stereoselectivity: no side L-products is detected in the DATA-catalyzed D-transamination. As in other PLP-dependent fold type IV transaminases, the functional unit of DATAs is a dimer. The active site is formed by amino acid residues of both subunits and binding of α-carboxylate group is crucial for proper substrate coordination. DATAs with promiscuous activity towards substrates without an α-carboxylate group, primary (R)-amines, have also been discovered and characterized. The promiscuous activity is achieved through the mobility of certain residues in the active site of DATAs. High stereoselectivity and stability of DATAs make then promising candidates for multienzyme cascade processes as biocatalysts of the (R)-stereoselective amination stage. Open configuration of active site makes binding and conversion of bulk non-natural substrates possible. The review describes in detail properties, structure, and relationships of DATAs from two currently known groups differing in organization of their active sites. The prospects for biotechnological applications of DATAs are discussed as well.

Molecular mechanisms underlying the green insect camouflage have puzzled researchers for over a century. Here, we isolated and identified a green water-soluble protein from the integument of bush-cricket Tettigonia cantans. De novo sequencing and cloning revealed a severely fragmented form of vitellogenins, ubiquitous and multifunctional, but still largely enigmatic glycolipoproteins essential for embryonic development and lacking structural characterization. The distinctive color of the identified chromoprotein results from binding of a remarkable combination of farnesylated bilins (recently identified, tentative heme A catabolites) and xanthophylls, which commensurably absorb light in the 600 to 700 nm and 400 to 550 nm spectral regions and thereby produce a hue that perfectly mimics foliage. The high-resolution crystal structure of this unique ~80 kDa dichromophoric protein, which we named "dibilinoxanthinin" (DBXN), revealed two DBXN protomers, each consisting of three polypeptides, with a novel fold enclosing a large hydrophobic cavity that accommodates two bilins, two luteins, and four phosphatidylcholines, all anchored by hydrogen bonds and giving DBXN unique biochemical and optical properties. Among the green insects tested, some contained yellow and blue chromophores in separate fractions, while others had green proteins similar to DBXN, although not necessarily of the same size. Surprisingly, we isolated and identified a larger vitellogenin proteoform with DBXN-like absorption, from the green huntsman spider Micrommata virescens. These data illustrate striking variations in the DBXN-related pigmentation mechanism among different green arthropods and suggest that vitellogenins may have undergone neofunctionalization, reflecting their potential for functional diversification.

Pyridoxal-5 '-phosphate '-phosphate (PLP)-dependent transaminases are key enzymes of amino acid metabolism in cells and remarkable biocatalysts of stereoselective amination for process chemistry applications. As cofactor-dependent enzymes, transaminases are prone to cofactor leakage. Here we discuss the holoenzyme-apoenzyme interconversion and the kinetics of PLP incorporation into the apo form of a PLP-dependent transaminase from Haliscomenobacter hydrossis. . PLP binding to the apoenzyme was slow in buffer, but was accelerated in the presence of substrates. Two crystal structures of the apoenzyme were obtained: the directly obtained apoenzyme (PDB ID: 7P8O) and the one obtained by soaking crystals of the holoenzyme in a phenylhydrazine solution (PDB ID: 8YRU). The mechanism of PLP association with the apoenzyme was proposed on the basis of structural analysis of these apo forms. Three rearrangement steps, including (I) anchoring of the PLP via the phosphate group, (II) displacement of two loops, and (III) Schiff-bonding between the PLP and the epsilon-amino group of the catalytic lysine residue, reconstituted the active holo form of the transaminase from H. hydrossis. . The results obtained allowed us to determine in the active site a permanent part and elements that are assembled by PLP, these findings may be useful for transaminase engineering for biocatalysis.

Cyanobacteria use soluble antenna megacomplexes, phycobilisomes (PBSs), to maximize light-harvesting efficiency and small photoswitchable orange carotenoid proteins (OCPs) to down-regulate PBSs in high light. Among known PBS morphologies, the one from the basal cyanobacterial genus Gloeobacter still lacks detailed structural characterization. Here, we reconstructed a cryo-electron microscopy structure of the >10-megadalton Gloeobacter violaceus PBS, with diverging, conformationally mobile bundles of rods composed of stacked phycoerythrin and phycocyanin hexamers, stemming from a pentacylindrical allophycocyanin core belted by auxiliary phycocyanin hexamers. We show how two Gloeobacter-specific multidomain linker proteins, Glr1262 and Glr2806, maintain this bundle-shaped architecture and reveal its differential regulation via nonphotochemical quenching by two OCP types of G. violaceus that recognize separate binding sites within the allophycocyanin core, including lateral cylinders absent in tricylindrical cores.

An interplay between the structural and physicochemical properties of the monoheme cytochromes of type c has been extensively studied. However new proteins belonging to this diverse family continue to reveal some novel and unique features. Here, we present the 1.15 Å structure of the low-potential cytochrome c546/556 from the bacterium Thioalkalivibrio paradoxus ARh1, which exhibits the prominent splitting of the Q bands in UV-visible spectra even at room temperature. The data obtained suggest that two conformations of the propionate 7 of the heme are responsible for the splitting of the Q bands. We propose that the degree of the splitting of the Q bands is correlated with the conformational lability of the heme propionates.

The CopC proteins are periplasmic copper binding proteins involved in bacterial copper homeostasis. One of the supposed functions of СopСs is to deliver copper ions for incorporation into the active sites of copper-containing oxidoreductases. In the sulfur oxidizing bacterium Thiolkalivibrio paradoxus, the genes encoding the CopCD proteins are located near the gene encoding the enzyme thiocyanate dehydrogenase (TcDH) containing three copper ions in the active site. The biochemical characterization demonstrated that CopC from Tv. paradoxus (tpCopC) has a high affinity for copper ions in both oxidation states (log KD = -16.3 ± 0.6 for Cu(II) and -11.1 ± 0.2 for Cu(I)). The protein Cu(II)-tpCopC forms a transient complex with TcDH, in which a copper ion could be transferred from tpCopC to the active site of TcDH. In the absence of a reducing agent, the transfer of 0.5 ± 0.2 copper ions is observed; under reducing conditions, the transfer of 2.4 ± 0.1 copper ions takes place followed by the activation of TcDH. Thus, CopC can act as a metallochaperone, providing the incorporation of copper ions into the active site of TcDH. The mechanism is proposed for the copper ion transfer from Cu(II)-tpCopC to TcDH through the intermediate reduction to form Cu(I)-tpCopC.

Understanding the structure-function relationships of pyridoxal-5'-phosphate (PLP)-dependent transaminases is key to advancing pyridoxal-phosphate-dependent catalysis and engineering transaminases for industrial applications. Despite our extensive knowledge of PLP-dependent enzymatic reactions, engineering transaminase activity and stability remains challenging. Here, we present the functional characterization of a novel PLP-dependent fold type IV transaminase from Desulfomonile tiedjei, alongside a detailed analysis of PLP binding and holoenzyme stability. This new transaminase exhibits activity toward various D-amino acids and (R)-phenylethylamine. Structural modeling and site-directed mutagenesis of residues in the second shell of the PLP-binding site revealed their roles in cofactor binding and the transaminase's catalytic efficiency. Notably, the T199Q variant demonstrated a fivefold increase in PLP affinity and improved activity under alkaline conditions. This is attributed to a newly formed hydrogen bond that stabilizes the N1-binding region of PLP. Glutamine at position 199 is not observed in homologous transaminases, making this non-natural substitution a novel and beneficial modification. These findings emphasize the importance of second-shell interactions in stabilizing PLP and expand our understanding of the structural diversity within PLP fold type IV transaminases. This paves the way for the engineering of more stable and versatile biocatalysts for industrial applications.

The LH2 complex is essential for light harvesting in many photosynthetic bacteria. To elucidate the specific structural role of carotenoids, we analyzed LH2 complexes from Ectothiorhodospira haloalkaliphila with inhibited carotenoid biosynthesis. This approach allowed us to study complexes incorporating the colorless carotenoid phytoene instead of the native, colored pigments. A 1.92 Å cryo‐EM reconstruction revealed that phytoene fully substitutes for the native carotenoids while maintaining the octameric symmetry of the complex and the precise arrangement of bacteriochlorophylls. These results demonstrate that the architectural function of carotenoids in LH2 complexes is maintained even when their light‐absorption capability is altered, providing new mechanistic insight into the structural basis of pigment–protein interactions in photosynthetic antenna complexes.

Structure-function relationships are key to understanding enzyme mechanisms, controlling enzyme activities, and designing biocatalysts. Here, we investigate the functions of arginine residues in the active sites of pyridoxal-5'-phosphate (PLP)-dependent non-canonical D-amino acid transaminases, focusing on the analysis of a transaminase from Haliscomenobacter hydrossis. Our results show that the tandem of arginine residues R28* and R90, which form the conserved R-[RK] motif in non-canonical D-amino acid transaminases, not only facilitates effective substrate binding but also regulates the catalytic properties of PLP. Non-covalent interactions between residues R28*, R90, and Y147 strengthen the hydrogen bond between Y147 and PLP, thereby maintaining the reactivity of the cofactor. Next, the R90 residue contributes to the stability of the holoenzyme. Finally, the R90I substitution induces structural changes that lead to substrate promiscuity, as evidenced by the effective binding of substrates with and without the α-carboxylate group. This study sheds light on the structural determinants of the activity of non-canonical D-amino acid transaminases. Understanding the structural basis of the active site plasticity in the non-canonical transaminase from H. hydrossis, which is characterized by effective conversion of D-amino acids and α-keto acids, may help to tailor it for industrial applications.

The work investigated the effect of four enzyme preparations (EP): Bacillolysin, Agroprot, Protozyme and Protozyme C (Russia), on smell, taste, as well as protein and peptide profiles of protein isolate isolated from Focor peas. It has been shown that enzyme treatment improves the odor characteristics of the isolate. Thus, it was possible to significantly reduce the severity of bean and herbal smell. At the same time, enzyme treatment also improved the taste of the isolate: it was possible to significantly reduce the severity of such disturbing flavors as legume, astringent, bitter and herbal. The results obtained made it possible to select EP (fungal acidic aspartate proteinase) to improve the organoleptic parameters of pea protein isolates intended for the manufacture of analogues of meat and dairy products.

The Orange Carotenoid Protein (OCP) is a unique photoreceptor crucial for cyanobacterial photoprotection. Best studied Synechocystis sp. PCC 6803 OCP belongs to the large OCP1 family. Downregulated by the Fluorescence Recovery Protein (FRP) in low-light, high-light-activated OCP1 binds to the phycobilisomes and performs non-photochemical quenching. Recently discovered families OCP2 and OCP3 remain structurally and functionally underexplored, and no systematic comparative studies have ever been conducted. Here we present two first crystal structures of OCP2 from morphoecophysiologically different cyanobacteria and provide their comprehensive structural, spectroscopic and functional comparison with OCP1, the recently described OCP3 and all-OCP ancestor. Structures enable correlation of spectroscopic signatures with the effective number of hydrogen and discovered here chalcogen bonds anchoring the ketocarotenoid in OCP, as well as with the rotation of the echinenone's β-ionone ring in the CTD. Structural data also helped rationalize the observed differences in OCP/FRP and OCP/phycobilisome functional interactions. These data are expected to foster OCP research and applications in optogenetics, targeted carotenoid delivery and cyanobacterial biomass engineering.

The effect of four enzyme preparations (EP), Bacillolysin, Agroprot, Protozyme, and Protozyme C (Russia), on the protein and peptide profiles of the protein isolate isolated from peas of the Focor variety, as well as on its smell and taste, was investigated in this work. It was shown that enzyme treatment can improve the odor characteristics of the isolate. Thus, it was possible to reduce significantly the severity of the bean and herbal smell. At the same time, enzyme treatment also improved the taste of the isolate: it was possible to reduce significantly the severity of disturbing flavors such as leguminous, astringent, bitter, and herbal. The results obtained allowed us to select EP (fungal acid aspartic protease) to improve the organoleptic parameters of pea protein isolates intended for the production of meat and dairy product analogs.

The effect of four enzyme preparations: bacillolysin, agroprot, protozyme and protozyme C (Russia) on solubility, emulsifying activity, emulsion stability, foaming and foam stability of isolates preparated from two varieties of peas was studied. It is shown that treatment with enzymes can increase the solubility of isolates at pH 5 by more than 7 times, the index of emulsifying activity at pH 5 by 1.5 to 2 times, and at pH 6 by almost 1.5 times; the stability index of the emulsion increased by about 20% at pH 5, and by 1.7 times (in one of the varieties) at pH 6; foaming increased by 2.4 to 3 times at pH 5, and at pH 6 by 1.8 to 3.7 times; foam stability increased by 25 to 33% at pH 5 and by more than 1.5 times (in one of the varieties) at pH 6. The results obtained made it possible to select an enzyme preparation (bacterial alkaline serine protease) to improve the parameters of pea protein isolates intended for the manufacture of analogues of fermented milk products.