Bright and stable monomeric green fluorescent protein derived from StayGold.

The high brightness and photostability of the green fluorescent protein StayGold make it a particularly attractive probe for long-term live-cell imaging; however, its dimeric nature precludes its application as a fluorescent tag for some proteins. Here, we report the development and crystal structures of a monomeric variant of StayGold, named mBaoJin, which preserves the beneficial properties of its precursor, while serving as a tag for structural proteins and membranes. Systematic benchmarking of mBaoJin against popular green fluorescent proteins and other recently introduced monomeric and pseudomonomeric derivatives of StayGold established mBaoJin as a bright and photostable fluorescent protein, exhibiting rapid maturation and high pH/chemical stability. mBaoJin was also demonstrated for super-resolution, long-term live-cell imaging and expansion microscopy. We further showed the applicability of mBaoJin for neuronal labeling in model organisms, including Caenorhabditis elegans and mice.

Expanded Substrate Specificity in D-Amino Acid Transaminases: A Case Study of Transaminase from Blastococcus saxobsidens.

Enzymes with expanded substrate specificity are good starting points for the design of biocatalysts for target reactions. However, the structural basis of the expanded substrate specificity is still elusive, especially in the superfamily of pyridoxal-5'-phosphate-dependent transaminases, which are characterized by a conserved organization of both the active site and functional dimer. Here, we analyze the structure-function relationships in a non-canonical D-amino acid transaminase from Blastococcus saxobsidens, which is active towards D-amino acids and primary (R)-amines. A detailed study of the enzyme includes a kinetic analysis of its substrate scope and a structural analysis of the holoenzyme and its complex with phenylhydrazine-a reversible inhibitor and analogue of (R)-1-phenylethylamine-a benchmark substrate of (R)-selective amine transaminases. We suggest that the features of the active site of transaminase from B. saxobsidens, such as the flexibility of the R34 and R96 residues, the lack of bulky residues in the ß-turn at the entrance to the active site, and the short O-pocket loop, facilitate the binding of substrates with and without α-carboxylate groups. The proposed structural determinants of the expanded substrate specificity can be used for the design of transaminases for the stereoselective amination of keto compounds.

In search for structural targets for engineering d-amino acid transaminase: modulation of pH optimum and substrate specificity.

The development of biocatalysts requires reorganization of the enzyme's active site to facilitate the productive binding of the target substrate and improve turnover number at desired conditions. Pyridoxal-5'-phosphate (PLP) - dependent transaminases are highly efficient biocatalysts for asymmetric amination of ketones and keto acids. However, transaminases, being stereoselective enzymes, have a narrow substrate specificity due to the ordered structure of the active site and work only in neutral-alkaline media. Here, we investigated the d-amino acid transaminase from Aminobacterium colombiense, with the active site organized differently from that of the canonical d-amino acid transaminase from Bacillus sp. YM-1. Using a combination of site-directed mutagenesis, kinetic analysis, molecular modeling, and structural analysis we determined the active site residues responsible for substrate binding, substrate differentiation, thermostability of a functional dimer, and affecting the pH optimum. We demonstrated that the high specificity toward d-glutamate/α-ketoglutarate is due to the interactions of a γ-carboxylate group with K237 residue, while binding of other substrates stems from the effectiveness of their accommodation in the active site optimized for d-glutamate/α-ketoglutarate binding. Furthermore, we showed that the K237A substitution shifts the catalytic activity optimum to acidic pH. Our findings are useful for achieving target substrate specificity and demonstrate the potential for developing and optimizing transaminases for various applications.

YTnC2, an improved genetically encoded green calcium indicator based on toadfish troponin C.

Genetically encoded calcium indicators based on truncated troponin C are attractive probes for calcium imaging due to their relatively small molecular size and twofold reduced calcium ion buffering. However, the best-suited members of this family, YTnC and cNTnC, suffer from low molecular brightness, limited dynamic range, and/or poor sensitivity to calcium transients in neurons. To overcome these limitations, we developed an enhanced version of YTnC, named YTnC2. Compared with YTnC, YTnC2 had 5.7-fold higher molecular brightness and 6.4-fold increased dynamic range in vitro. YTnC2 was successfully used to reveal calcium transients in the cytosol and in the lumen of mitochondria of both mammalian cells and cultured neurons. Finally, we obtained and analyzed the crystal structure of the fluorescent domain of the YTnC2 mutant.

Mechanism of D-Cycloserine Inhibition of D-Amino Acid Transaminase from Haliscomenobacter hydrossis.

D-cycloserine inhibits pyridoxal-5'-phosphate (PLP)-dependent enzymes. Inhibition effect depend on organization of the active site and mechanism of the catalyzed reaction. D-cycloserine interacts with the PLP form of the enzyme similarly to the substrate (amino acid), and this interaction is predominantly reversible. Several products of the interaction of PLP with D-cycloserine are known. For some enzymes formation of a stable aromatic product - hydroxyisoxazole-pyridoxamine-5'-phosphate at certain pH - leads to irreversible inhibition. The aim of this work was to study the mechanism of D-cycloserine inhibition of the PLP-dependent D-amino acid transaminase from Haliscomenobacter hydrossis. Spectral methods revealed several products of interaction of D-cycloserine with PLP in the active site of transaminase: oxime between PLP and ß-aminooxy-D-alanine, ketimine between pyridoxamine-5'-phosphate and cyclic form of D-cycloserine, and pyridoxamine-5'-phosphate. Formation of hydroxyisoxazole-pyridoxamine-5'-phosphate was not observed. 3D structure of the complex with D-cycloserine was obtained using X-ray diffraction analysis. In the active site of transaminase, a ketimine adduct between pyridoxamine-5'-phosphate and D-cycloserine in the cyclic form was found. Ketimine occupied two positions interacting with different active site residues via hydrogen bonds. Using kinetic and spectral methods we have shown that D-cycloserine inhibition is reversible, and activity of the inhibited transaminase from H. hydrossis could be restored by adding excess of keto substrate or excess of cofactor. The obtained results confirm reversibility of the inhibition by D-cycloserine and interconversion of various adducts of D-cycloserine and PLP.

FNCaMP, ratiometric green calcium indicator based on mNeonGreen protein.

Neurobiologists widely use green genetically encoded calcium indicators (GECIs) for visualization of neuronal activity. Among them, ratiometric GECIs allow imaging of both active and non-active neuronal populations. However, they are not popular, since their properties are inferior to intensiometric GCaMP series of GECIs. The most characterized and developed ratiometric green GECI is FGCaMP7. However, the dynamic range and sensitivity of its large Stock's shift green (LSS-Green) form is significantly lower than its Green form and its molecular design is not optimal. To address these drawbacks, we engineered a ratiometric green calcium indicator, called FNCaMP, which is based on bright mNeonGreen protein and calmodulin from A. niger and has optimal NTnC-like design. We compared the properties of the FNCaMP and FGCaMP7 indicators in vitro, in mammalian cells, and in neuronal cultures. Finally, we obtained and analyzed X-ray structure of the FNCaMP indicator.

Friunavirus Phage-Encoded Depolymerases Specific to Different Capsular Types of Acinetobacter baumannii.

Acinetobacter baumannii is a critical priority nosocomial pathogen that produces a variety of capsular polysaccharides (CPSs), the primary receptors for specific depolymerase-carrying phages. In this study, the tailspike depolymerases (TSDs) encoded in genomes of six novel Friunaviruses, APK09, APK14, APK16, APK86, APK127v, APK128, and one previously described Friunavirus phage, APK37.1, were characterized. For all TSDs, the mechanism of specific cleavage of corresponding A. baumannii capsular polysaccharides (CPSs) was established. The structures of oligosaccharide fragments derived from K9, K14, K16, K37/K3-v1, K86, K127, and K128 CPSs degradation by the recombinant depolymerases have been determined. The crystal structures of three of the studied TSDs were obtained. A significant reduction in mortality of Galleria mellonella larvae infected with A. baumannii of K9 capsular type was shown in the example of recombinant TSD APK09_gp48. The data obtained will provide a better understanding of the interaction of phage-bacterial host systems and will contribute to the formation of principles of rational usage of lytic phages and phage-derived enzymes as antibacterial agents.

Combined Structural and Computational Study of the mRubyFT Fluorescent Timer Locked in Its Blue Form.

The mRubyFT is a monomeric genetically encoded fluorescent timer based on the mRuby2 fluorescent protein, which is characterized by the complete maturation of the blue form with the subsequent conversion to the red one. It has higher brightness in mammalian cells and higher photostability compared with other fluorescent timers. A high-resolution structure is a known characteristic of the mRubyFT with the red form chromophore, but structural details of its blue form remain obscure. In order to obtain insight into this, we obtained an S148I variant of the mRubyFT (mRubyFTS148I) with the blocked over time blue form of the chromophore. X-ray data at a 1.8 Å resolution allowed us to propose a chromophore conformation and its interactions with the neighboring residues. The imidazolidinone moiety of the chromophore is completely matured, being a conjugated π-system. The methine bridge is not oxidized in the blue form bringing flexibility to the phenolic moiety that manifests itself in poor electron density. Integration of these data with the results of molecular dynamic simulation disclosed that the OH group of the phenolic moiety forms a hydrogen bond with the side chain of the T163 residue. A detailed comparison of mRubyFTS148I with other available structures of the blue form of fluorescent proteins, Blue102 and mTagBFP, revealed a number of characteristic differences. Molecular dynamic simulations with the combined quantum mechanic/molecular mechanic potentials demonstrated that the blue form exists in two protonation states, anion and zwitterion, both sharing enolate tautomeric forms of the C=C-O- fragment. These two forms have similar excitation energies, as evaluated by calculations. Finally, excited state molecular dynamic simulations showed that excitation of the chromophore in both protonation states leads to the same anionic fluorescent state. The data obtained shed light on the structural features and spectral properties of the blue form of the mRubyFT timer.

To the Understanding of Catalysis by D-Amino Acid Transaminases: A Case Study of the Enzyme from Aminobacterium colombiense.

Pyridoxal-5'-phosphate (PLP)-dependent transaminases are highly efficient biocatalysts for stereoselective amination. D-amino acid transaminases can catalyze stereoselective transamination producing optically pure D-amino acids. The knowledge of substrate binding mode and substrate differentiation mechanism in D-amino acid transaminases comes down to the analysis of the transaminase from Bacillus subtilis. However, at least two groups of D-amino acid transaminases differing in the active site organization are known today. Here, we present a detailed study of D-amino acid transaminase from the gram-negative bacterium Aminobacterium colombiense with a substrate binding mode different from that for the transaminase from B. subtilis. We study the enzyme using kinetic analysis, molecular modeling, and structural analysis of holoenzyme and its complex with D-glutamate. We compare the multipoint binding of D-glutamate with the binding of other substrates, D-aspartate and D-ornithine. QM/MM MD simulation reveals that the substrate can act as a base and its proton can be transferred from the amino group to the α-carboxylate group. This process occurs simultaneously with the nucleophilic attack of the PLP carbon atom by the nitrogen atom of the substrate forming gem-diamine at the transimination step. This explains the absence of the catalytic activity toward (R)-amines that lack an α-carboxylate group. The obtained results clarify another substrate binding mode in D-amino acid transaminases and underpinned the substrate activation mechanism.

Crystal Structure of Inhibitor-Bound Bacterial Oligopeptidase B in the Closed State: Similarity and Difference between Protozoan and Bacterial Enzymes.

The crystal structure of bacterial oligopeptidase B from Serratia proteamaculans (SpOpB) in complex with a chloromethyl ketone inhibitor was determined at 2.2 Å resolution. SpOpB was crystallized in a closed (catalytically active) conformation. A single inhibitor molecule bound simultaneously to the catalytic residues S532 and H652 mimicked a tetrahedral intermediate of the catalytic reaction. A comparative analysis of the obtained structure and the structure of OpB from Trypanosoma brucei (TbOpB) in a closed conformation showed that in both enzymes, the stabilization of the D-loop (carrying the catalytic D) in a position favorable for the formation of a tetrahedral complex occurs due to interaction with the neighboring loop from the ß-propeller. However, the modes of interdomain interactions were significantly different for bacterial and protozoan OpBs. Instead of a salt bridge (as in TbOpB), in SpOpB, a pair of polar residues following the catalytic D617 and a pair of neighboring arginine residues from the ß-propeller domain formed complementary oppositely charged surfaces. Bioinformatics analysis and structural modeling show that all bacterial OpBs can be divided into two large groups according to these two modes of D-loop stabilization in closed conformations.

Blue-to-Red TagFT, mTagFT, mTsFT, and Green-to-FarRed mNeptusFT2 Proteins, Genetically Encoded True and Tandem Fluorescent Timers.

True genetically encoded monomeric fluorescent timers (tFTs) change their fluorescent color as a result of the complete transition of the blue form into the red form over time. Tandem FTs (tdFTs) change their color as a consequence of the fast and slow independent maturation of two forms with different colors. However, tFTs are limited to derivatives of the mCherry and mRuby red fluorescent proteins and have low brightness and photostability. The number of tdFTs is also limited, and there are no blue-to-red or green-to-far-red tdFTs. tFTs and tdFTs have not previously been directly compared. Here, we engineered novel blue-to-red tFTs, called TagFT and mTagFT, which were derived from the TagRFP protein. The main spectral and timing characteristics of the TagFT and mTagFT timers were determined in vitro. The brightnesses and photoconversions of the TagFT and mTagFT tFTs were characterized in live mammalian cells. The engineered split version of the TagFT timer matured in mammalian cells at 37 °C and allowed the detection of interactions between two proteins. The TagFT timer under the control of the minimal arc promoter, successfully visualized immediate-early gene induction in neuronal cultures. We also developed and optimized green-to-far-red and blue-to-red tdFTs, named mNeptusFT and mTsFT, which were based on mNeptune-sfGFP and mTagBFP2-mScarlet fusion proteins, respectively. We developed the FucciFT2 system based on the TagFT-hCdt1-100/mNeptusFT2-hGeminin combination, which could visualize the transitions between the G1 and S/G2/M phases of the cell cycle with better resolution than the conventional Fucci system because of the fluorescent color changes of the timers over time in different phases of the cell cycle. Finally, we determined the X-ray crystal structure of the mTagFT timer and analyzed it using directed mutagenesis.

cNTnC and fYTnC2, Genetically Encoded Green Calcium Indicators Based on Troponin C from Fast Animals.

NTnC-like green fluorescent genetically encoded calcium indicators (GECIs) with two calcium ion binding sites were constructed using the insertion of truncated troponin C (TnC) from Opsanus tau into green fluorescent proteins (GFPs). These GECIs are small proteins containing the N- and C-termini of GFP; they exert a limited effect on the cellular free calcium ion concentration; and in contrast to calmodulin-based calcium indicators they lack undesired interactions with intracellular proteins in neurons. The available TnC-based NTnC or YTnC GECIs had either an inverted response and high brightness but a limited dynamic range or a positive response and fast kinetics in neurons but lower brightness and an enhanced but still limited dF/F dynamic range. Here, we solved the crystal structure of NTnC at 2.5 Å resolution. Based on this structure, we developed positive NTnC2 and inverted iNTnC2 GECIs with a large dF/F dynamic range in vitro but very slow rise and decay kinetics in neurons. To overcome their slow responsiveness, we swapped TnC from O. tau in NTnC2 with truncated troponin C proteins from the muscles of fast animals, namely, the falcon, hummingbird, cheetah, bat, rattlesnake, and ant, and then optimized the resulting constructs using directed molecular evolution. Characterization of the engineered variants using purified proteins, mammalian cells, and neuronal cultures revealed cNTnC GECI with truncated TnC from Calypte anna (hummingbird) to have the largest dF/F fluorescence response and fast dissociation kinetics in neuronal cultures. In addition, based on the insertion of truncated TnCs from fast animals into YTnC2, we developed fYTnC2 GECI with TnC from Falco peregrinus (falcon). The purified proteins cNTnC and fYTnC2 had 8- and 6-fold higher molecular brightness and 7- and 6-fold larger dF/F responses to the increase in Ca2+ ion concentration than YTnC, respectively. cNTnC GECI was also 4-fold more photostable than YTnC and fYTnC2 GECIs. Finally, we assessed the developed GECIs in primary mouse neuronal cultures stimulated with an external electric field; in these conditions, cNTnC had a 2.4-fold higher dF/F fluorescence response than YTnC and fYTnC2 and was the same or slightly slower (1.4-fold) than fYTnC2 and YTnC in the rise and decay half-times, respectively.

Structure of the DNMT3B ADD domain suggests the absence of a DNMT3A-like autoinhibitory mechanism.

De novo DNA methylation in early mammalian development depends on the activity of the DNMT3 methyltransferase family. An autoinhibitory mechanism involving the interaction between ADD and the catalytic domains of DNMT3A has been described. ADD is a zinc-coordinating histone-binding domain. The ADD domain of DNMT3A, when bound to a K4-unmethylated histone H3 tail, switches the enzyme to its catalytically active state. DNMT3B is another de novo methyltransferase enzyme with a more strict tissue- and stage-specific expression profile and a slightly different site specificity, lacking cooperative DNA methylation activity. Here, we obtained the crystal structure of the DNMT3B ADD domain, which demonstrated the extended conformation of the autoinhibitory loop even in the absence of the histone H3 tail. The lack of interaction between DNMT3B ADD and the methyltransferase domain was confirmed using an in vitro pull-down assay. The structural rearrangements in the loop also created an additional protein interaction interface leading to the formation of trimers in crystal, which may reflect their possible involvement in some unknown protein-protein interactions. Our results suggest that DNMT3B, in contrast to DNMT3A, has different modes of regulation of its activity that are independent of H3K4 methylation status.

Structural insights into highly similar spatial organization of zinc-finger associated domains with a very low sequence similarity.

ZAD is a C4 zinc-coordinating domain often found at the N-terminus mostly of arthropodan transcription factors with multiple C2H2 zinc-finger domains involved in the regulation of chromosome architecture and promotor activity. ZADs predominantly form homodimers and have low primary sequence similarity. We obtained three crystal structures of the most phylogenetically distant Drosophila ZADs and structure of the only known ZAD-like domain from a mammalian protein (ZNF276). All ZAD structures demonstrate unity of the spatial fold as well as some unique structural features. The specific homodimerization of ZAD is primarily determined by the position and size of secondary structural elements and is further strengthened by a number of unique interactions between subunits. Structural comparison allowed for unraveling key sequence features underlying the similarity of the spatial fold. These features result in a broad variety of ZADs in Arthropod C2H2 proteins, allowing for the emergence of a wide range of highly specific homodimers.

The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent Timer.

Genetically encoded monomeric blue-to-red fluorescent timers (mFTs) change their fluorescent color over time. mCherry-derived mFTs were used for the tracking of the protein age, visualization of the protein trafficking, and labeling of engram cells. However, the brightness of the blue and red forms of mFTs are 2-3- and 5-7-fold dimmer compared to the brightness of the enhanced green fluorescent protein (EGFP). To address this limitation, we developed a blue-to-red fluorescent timer, named mRubyFT, derived from the bright mRuby2 red fluorescent protein. The blue form of mRubyFT reached its maximum at 5.7 h and completely transformed into the red form that had a maturation half-time of 15 h. Blue and red forms of purified mRubyFT were 4.1-fold brighter and 1.3-fold dimmer than the respective forms of the mCherry-derived Fast-FT timer in vitro. When expressed in mammalian cells, both forms of mRubyFT were 1.3-fold brighter than the respective forms of Fast-FT. The violet light-induced blue-to-red photoconversion was 4.2-fold less efficient in the case of mRubyFT timer compared to the same photoconversion of the Fast-FT timer. The timer behavior of mRubyFT was confirmed in mammalian cells. The monomeric properties of mRubyFT allowed the labeling and confocal imaging of cytoskeleton proteins in live mammalian cells. The X-ray structure of the red form of mRubyFT at 1.5 Å resolution was obtained and analyzed. The role of the residues from the chromophore surrounding was studied using site-directed mutagenesis.

Unusual substrate specificity in GH family 12: structure-function analysis of glucanases Bgh12A and Xgh12B from Aspergillus cervinus, and Egh12 from Thielavia terrestris.

In this study, we compared the properties and structures of three fungal GH12 enzymes: the strict endoglucanase Bgh12A and the xyloglucanase Xgh12B from Aspergillus cervinus, and the endoglucanase Egh12 from Thielavia terrestris combining activity on linear ß-glucan and branched xyloglucan. Egh12 from T. terrestris was produced in Pichia pastoris, purified, and characterized as a thermostable enzyme with maximal activity at 70 ºC and a half-life time of 138 min at 65 °C. We for the first time demonstrated that the GH12 endoglucanases Egh12 and Bgh12A, but not the strict xyloglucanase Xgh12B, hydrolyzed (1,3)-ß-linkages in (1,3;1,4)-ß-D-glucooligosaccharides and had transglycosylase activity on (1,3)-ß-D-glucooligosaccharides. Phylogenetic analysis indicated that Egh12 from T. terrestris and Bgh12A from A. cervinus are more related than Bgh12A and Xgh12B isolated from one strain. The X-ray structure of Bgh12A was determined with 2.17 Å resolution and compared with 3D-homology models of Egh12 and Xgh12B. The enzymes have a ß-jelly roll structure with a catalytic cleft running across the protein. Comparative analysis and a docking study demonstrated the importance of endoglucanase-specific loop 1 partly covering the catalytic cleft for correct placement of the linear substrates. Variability in substrate specificity between the GH12 endoglucanases is determined by non-conservative residues in structural loops framing the catalytic cleft. A residue responsible for the thermostability of Egh12 was predicted. The key structural elements and residues described in this study may serve as potential targets for modification aimed at the improvement of enzymatic properties. KEY POINTS: • Thermostable endoglucanase Egh12 from T. terrestris was produced in P. pastoris, purified, and characterized • The X-ray structure of GH12 endoglucanase Bgh12A from A. cervinus was resolved • GH12 endoglucanases, but not GH12 xyloglucanases, hydrolyze (1,3)-ß-linkages in (1,3;1,4)-ß-D-glucooligosaccharides.

LSSmScarlet, dCyRFP2s, dCyOFP2s and CRISPRed2s, Genetically Encoded Red Fluorescent Proteins with a Large Stokes Shift.

Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the right LSSRFP for a given application is hampered by the lack of a side-by-side comparison of the LSSRFPs' performance. In this study, we employed rational design and random mutagenesis to convert conventional bright RFP mScarlet into LSSRFP, called LSSmScarlet, characterized by excitation/emission maxima at 470/598 nm. In addition, we utilized the previously reported LSSRFPs mCyRFP1, CyOFP1, and mCRISPRed as templates for directed molecular evolution to develop their optimized versions, called dCyRFP2s, dCyOFP2s and CRISPRed2s. We performed a quantitative assessment of the developed LSSRFPs and their precursors in vitro on purified proteins and compared their brightness at 488 nm excitation in the mammalian cells. The monomeric LSSmScarlet protein was successfully utilized for the confocal imaging of the structural proteins in live mammalian cells and multicolor confocal imaging in conjugation with other FPs. LSSmScarlet was successfully applied for dual-color two-photon imaging in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet protein at the resolution of 1.4 Å that revealed a hydrogen bond network supporting excited-state proton transfer (ESPT). Quantum mechanics/molecular mechanics molecular dynamic simulations confirmed the ESPT mechanism of a large Stokes shift. Structure-guided mutagenesis revealed the role of R198 residue in ESPT that allowed us to generate a variant with improved pH stability. Finally, we showed that LSSmScarlet protein is not appropriate for STED microscopy as a consequence of LSSRed-to-Red photoconversion with high-power 775 nm depletion light.

Mechanisms of CP190 Interaction with Architectural Proteins in Drosophila Melanogaster.

Most of the known Drosophila architectural proteins interact with an important cofactor, CP190, that contains three domains (BTB, M, and D) that are involved in protein-protein interactions. The highly conserved N-terminal CP190 BTB domain forms a stable homodimer that interacts with unstructured regions in the three best-characterized architectural proteins: dCTCF, Su(Hw), and Pita. Here, we identified two new CP190 partners, CG4730 and CG31365, that interact with the BTB domain. The CP190 BTB resembles the previously characterized human BCL6 BTB domain, which uses its hydrophobic groove to specifically associate with unstructured regions of several transcriptional repressors. Using GST pull-down and yeast two-hybrid assays, we demonstrated that mutations in the hydrophobic groove strongly affect the affinity of CP190 BTB for the architectural proteins. In the yeast two-hybrid assay, we found that architectural proteins use various mechanisms to improve the efficiency of interaction with CP190. Pita and Su(Hw) have two unstructured regions that appear to simultaneously interact with hydrophobic grooves in the BTB dimer. In dCTCF and CG31365, two adjacent regions interact simultaneously with the hydrophobic groove of the BTB and the M domain of CP190. Finally, CG4730 interacts with the BTB, M, and D domains of CP190 simultaneously. These results suggest that architectural proteins use different mechanisms to increase the efficiency of interaction with CP190.

The Uncommon Active Site of D-Amino Acid Transaminase from Haliscomenobacter hydrossis: Biochemical and Structural Insights into the New Enzyme.

Among industrially important pyridoxal-5'-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from Haliscomenobacter hydrossis (Halhy). The new enzyme is strictly specific towards D-amino acids and their keto analogs; it demonstrates one of the highest rates of transamination between D-glutamate and pyruvate. We obtained the crystal structure of the Halhy in the holo form with the protonated Schiff base formed by the K143 and the PLP. Structural analysis revealed a novel set of the active site residues that differ from the key residues forming the active sites of the previously studied D-amino acids transaminases. The active site of Halhy includes three arginine residues, one of which is unique among studied transaminases. We identified critical residues for the Halhy catalytic activity and suggested functions of the arginine residues based on the comparative structural analysis, mutagenesis, and molecular modeling simulations. We suggested a strong positive charge in the O-pocket and the unshaped P-pocket as a structural code for the D-amino acid specificity among transaminases of PLP fold type IV. Characteristics of Halhy complement our knowledge of the structural basis of substrate specificity of D-amino acid transaminases and the sequence-structure-function relationships in these enzymes.

Probing the role of the residues in the active site of the transaminase from Thermobaculum terrenum.

Creating biocatalysts for (R)-selective amination effectively is highly desirable in organic synthesis. Despite noticeable progress in the engineering of (R)-amine activity in pyridoxal-5'-phosphate-dependent transaminases of fold type IV, the specialization of the activity is still an intuitive task, as there is poor understanding of sequence-structure-function relationships. In this study, we analyzed this relationship in transaminase from Thermobaculum terrenum, distinguished by expanded substrate specificity and activity in reactions with L-amino acids and (R)-(+)-1-phenylethylamine using α-ketoglutarate and pyruvate as amino acceptors. We performed site-directed mutagenesis to create a panel of the enzyme variants, which differ in the active site residues from the parent enzyme to a putative transaminase specific to (R)-primary amines. The variants were examined in the overall transamination reactions and half-reaction with (R)-(+)-1-phenylethylamine. A structural analysis of the most prominent variants revealed a spatial reorganization in the active sites, which caused changes in activity. Although the specialization to (R)-amine transaminase was not implemented, we succeeded in understanding the role of the particular active site residues in expanding substrate specificity of the enzyme. We showed that the specificity for (R)-(+)-1-phenylethylamine in transaminase from T. terrenum arises without sacrificing the specificity for L-amino acids and α-ketoglutarate and in consensus with it.