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Glycans exhibit significant structural diversity due to the flexibility of glycosidic bonds linking their constituent monosaccharides and the formation of numerous hydrogen bonds. The present work searches a simulated ensemble of glycan chain conformations attached to the catalytic domain of N-glycosylated human carbonic anhydrase IX (HCA IX-c) to identify conformations pointed away or back-folded toward the protein surface guided by different amino acid residues. A series of classical molecular dynamics (MD) simulation studies for a total of 30 μs followed by accelerated MD simulations for a total of 2 μs have been performed using two different force fields to capture varying degrees of fluctuations of both glycan chain and HCA IX. From the underlying free energy profile and kinetics derived using hidden Markov state model, several stable glycan orientations are identified that extend away from the protein surface and convert among each other with rate constants of the order 10 7 -10 10 S -1 . Most importantly, we have identified a rare glycan conformation which reaches close to a catalytically important amino acid residue, Glu-106. We further enlist the protein residues that couple such less frequent event of the glycan chain back-folding toward the surface of the protein.

Protein phosphatase 1D (PPM1D, Wip1) is induced by the tumor suppressor p53 during DNA damage response signaling and acts as an oncoprotein in several human cancers. Although PPM1D is a potential therapeutic target, insights into its atomic structure were challenging due to flexible regions unique to this family member. Here, we report the first crystal structure of the PPM1D catalytic domain to 1.8 Å resolution. The structure reveals the active site with two Mg 2+ ions bound, similar to other structures. The flap subdomain and B-loop, which are crucial for substrate recognition and catalysis, were also resolved, with the flap forming two short helices and three short β-strands that are followed by an irregular loop. Unexpectedly, a nitrogen-oxygen-sulfur bridge was identified in the catalytic domain. Molecular dynamics simulations and kinetic studies provided further mechanistic insights into the regulation of PPM1D catalytic activity. In particular, the kinetic experiments demonstrated a magnesium concentration-dependent lag in PPM1D attaining steady-state velocity, a feature of hysteretic enzymes that show slow transitions compared with catalytic turnover. All combined, these results advance the understanding of PPM1D function and will support the development of PPM1D-targeted therapeutics., Competing Interests: Conflicts of interests The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

Many ATP-binding cassette transporters are regulated by phosphorylation on long and disordered loops which presents a challenge to visualize with structural methods. We have trapped an activated state of the regulatory domain (R-domain) of yeast cadmium factor 1 (Ycf1) by enzymatically enriching the phosphorylated state. A 3.23 Å cryo-EM structure reveals an R-domain structure with four phosphorylated residues and the position for the entire R-domain. The structure reveals key R-domain interactions including a bridging interaction between NBD1 and NBD2 and an interaction with the R-insertion, another regulatory region. We scanned these interactions by systematically replacing segments along the entire R-domain with scrambled combinations of alanine, glycine, and glutamine and probing function under cellular conditions that require the Ycf1 function. We find a close match with these interactions and interacting regions on our R-domain structure that points to the importance of most well-structured segments for function. We propose a model where the R-domain stabilizes a transport-competent state upon phosphorylation by enveloping NBD1 entirely., (© 2024 Souza Amado de Carvalho et al.)

Cyclin-dependent kinase-like 5 (CDKL5) is a serine/threonine protein kinase involved in human brain development and functioning. Mutations in CDKL5, especially in its catalytic domain, cause a severe developmental condition named CDKL5 deficiency disorder. Nevertheless, molecular studies investigating the structural consequences of such mutations are still missing. The CDKL5 catalytic domain harbors different sites of post-translational modification, such as phosphorylations, but their role in catalytic activity, protein folding, and stability has not been entirely investigated. With this work, we describe the expression pattern of the CDKL5 catalytic domain in Escherichia coli demonstrating that it predominantly aggregates. However, the use of solubility tags, the lowering of the expression temperature, the manual codon optimization to overcome an internal translational start, and the incubation of the protein with K + and MgATP allow the collection of a soluble catalytically active kinase. Interestingly, the resulting protein exhibits hypophosphorylation compared to its eukaryotic counterpart, proving that bacteria are a useful tool to achieve almost unmodified CDKL5. Posing questions about the CDKL5 autoactivation mechanism and the determinants for its stability, this research provides a valuable platform for comparative biophysical studies between bacterial and eukaryotic-expressed proteins, contributing to our understanding of neurodevelopmental disorders associated with CDKL5 dysfunction.

Considering the structure of the bacterial GH15 family glucoamylase (GA), Thermoplasma trehalase Tvn1315 may be composed of a β-sandwich domain (BD) and a catalytic domain (CD). Tvn1315 BD weakly binds to insoluble β-glucans, such as cellulose, and helps fold CD. To determine how aromatic residues contribute to proper folding and enzyme activity, we performed alanine scanning for 32 aromatic residues in the BD. The study did not identify a single residue involved in glucan binding. However, several aromatic residues were found to be involved in BD or CD folding and in modulating the activity of the full-length enzyme. Among those aromatic residue mutations, the W43A mutation led to reduced solubility of the BD and full-length protein and resulted in a full-length enzyme with significantly lower activity. The activity of W43F and W43Y was significantly higher than that of W43A. In addition, Ala substitutions of Tyr83, Tyr113, and Tyr17 led to a reduction in trehalase activity, but Phe substitutions of these residues could be tolerated, as these mutants maintained activities similar to WT activity. Thus, these aromatic residues in BD may interact with CD and modulate enzyme activity. KEY POINTS: • Aromatic residues in the BD are involved in BD and CD folding. • Aromatic residues in the BD near the CD active site modulate enzyme activity. • BD interacts with CD and closely modulates enzyme activity., (© 2024. The Author(s).)

In order to obtain α-glucan phosphorylase with good heterologous expression level and enzymatic properties and to investigate the impact of structural domains on the enzyme, a novel α-glucan phosphorylase TsGP was screened from the hyperthermophilic archaeon Thermococcus sp. EP1 through database mining and sequence analysis. After confirming that the C-terminal of TsGP was a non-catalytic domain in the three-dimensional structure predicted by AlphaFold2, a C-terminal truncated mutant ΔTsGP was constructed. Heterologous recombinant expression of TsGP and ΔTsGP was performed in E.coli BL21(DE3), and their enzymatic properties were characterized. The results showed that the expression level of ΔTsGP in E.coli was 3.76 times higher than that of TsGP. At the optimal reaction temperature 70 ℃, the specific enzyme activity of ΔTsGP was 23.87 U/mg, which was 1.3 times higher than that of TsGP. Under 50~65 ℃ which was widely applied as industrial catalytic temperature, the thermal stability of ΔTsGP was equivalent to that of TsGP, and the enzyme activity was higher. In addition, ΔTsGP and TsGP had the same substrate specificity, their smallest substrate was maltotriose, and as the substrate chain length increased, their specific enzyme activity gradually increased. Using maltoheptaose as the substrate, the kcat value of ΔTsGP was 37.09 s−1, which was 1.21 times higher than that of TsGP, but its substrate affinity (Km=3.30 mmol/L) was reduced by 2.77 times. Through structural analysis and non-catalytic domain truncation strategies, the study successfully improved the expression level of TsGP in E. coli and the specific enzyme activity under industrial enzyme catalytic conditions. The study would provide guidance for the efficient expression and application of αGPase protein and lays the foundation for further optimizing the performance of this enzyme through engineering modification.

APOBEC3G, part of the AID/APOBEC cytidine deaminase family, is crucial for antiviral immunity. It has two zinc-coordinated cytidine-deaminase domains. The non-catalytic N-terminal domain strongly binds to nucleic acids, whereas the C-terminal domain catalyzes C-to-U editing in single-stranded DNA. The interplay between the two domains is not fully understood. Here, we show that DNA editing function of rhesus macaque APOBEC3G on linear and hairpin loop DNA is enhanced by AA or GA dinucleotide motifs present downstream in the 3'-direction of the target-C editing sites. The effective distance between AA/GA and the target-C sites is contingent on the local DNA secondary structure. We present two co-crystal structures of rhesus macaque APOBEC3G bound to ssDNA containing AA and GA, revealing the contribution of the non-catalytic domain in capturing AA/GA DNA. Our findings elucidate the molecular mechanism of APOBEC3G's cooperative function, which is critical for its antiviral role and its contribution to mutations in cancer genomes., (© 2024. The Author(s).)

Abstract APOBEC3G, part of the AID/APOBEC cytidine deaminase family, is crucial for antiviral immunity. It has two zinc-coordinated cytidine-deaminase domains. The non-catalytic N-terminal domain strongly binds to nucleic acids, whereas the C-terminal domain catalyzes C-to-U editing in single-stranded DNA. The interplay between the two domains is not fully understood. Here, we show that DNA editing function of rhesus macaque APOBEC3G on linear and hairpin loop DNA is enhanced by AA or GA dinucleotide motifs present downstream in the 3’-direction of the target-C editing sites. The effective distance between AA/GA and the target-C sites is contingent on the local DNA secondary structure. We present two co-crystal structures of rhesus macaque APOBEC3G bound to ssDNA containing AA and GA, revealing the contribution of the non-catalytic domain in capturing AA/GA DNA. Our findings elucidate the molecular mechanism of APOBEC3G’s cooperative function, which is critical for its antiviral role and its contribution to mutations in cancer genomes.

The L-gulonolactone oxidase enzyme (GULO) catalyzes the last step of L-ascorbic acid (vitamin C) biosynthesis. This enzymatic activity is lost in primates. The full-length rat GULO has been previously produced in plants and demonstrated to be active. In this study, we compared the activity of two variants of GULO produced in Escheriachia coli cells, full-length rat GULO (fGULO) and its C-terminal catalytic domain (cGULO). The expression and purification of the recombinant proteins were optimized, and their biological activity was confirmed by two methods, the GULO activity assay in the protein extracts and the ‘in-gel’ staining for GULO activity. Both variants of recombinant GULO were biologically active in both assays. However, cGULO is more promising than fGULO for ascorbic acid production because it is more efficiently produced by bacteria. Furthermore, the optimal activities of the fGULO and cGULO recombinant proteins were observed at pH 7 and 6.5, and at temperatures of 40 and 30 °C, respectively. Kinetic studies revealed that at low substrate concentrations, Km values for fGULO and cGULO were 53.5 ± 5 and 42 ± 6.3 µM, respectively.

Recent research certified that DOT1L and its mutations represented by R231Q were potential targets for the treatment of lung cancer. Herein, a series of adenosine-containing derivatives were identified with DOT1LR231Q inhibition through antiproliferation assay and Western blot analysis in the H460R231Q cell. The most promising compound 37 significantly reduced DOT1LR231Q mediated H3K79 methylation and effectively inhibited the proliferation, self-renewal, migration, and invasion of lung cancer cell lines at low micromolar concentrations. The cell permeability and cellular target engagement of 37 were verified by both CETSA and DARTS assays. In the H460R231Q OE cell-derived xenograft (CDX) model, 37 displayed pronounced tumor growth inhibition after intraperitoneal administration at 20 mg/kg dose for 3 weeks (TGI = 54.38%), without obvious toxicities. A pharmacokinetic study revealed that 37 possessed tolerable properties (t1/2 = 1.93 ± 0.91 h, F = 97.2%) after intraperitoneal administration in rats. Mechanism study confirmed that 37 suppressed malignant phenotypes of lung cancer carrying R231Q gain-of-function mutation via the MAPK/ERK signaling pathway. Moreover, analysis of the binding modes between molecules and DOT1LWT/R231Q proteins put forward the “Induced-fit” allosteric model in favor to the discovery of potent DOT1L candidates.

Tyrosyl DNA phosphodiesterase 1 (TDP1) and DNA Ligase IIIα (LigIIIα) are key enzymes in single-strand break (SSB) repair. TDP1 removes 3'-tyrosine residues remaining after degradation of DNA topoisomerase (TOP) 1 cleavage complexes trapped by either DNA lesions or TOP1 inhibitors. It is not known how TDP1 is linked to subsequent processing and LigIIIα-catalyzed joining of the SSB. Here we define a direct interaction between the TDP1 catalytic domain and the LigIII DNA-binding domain (DBD) regulated by conformational changes in the unstructured TDP1 N-terminal region induced by phosphorylation and/or alterations in amino acid sequence. Full-length and N-terminally truncated TDP1 are more effective at correcting SSB repair defects in TDP1 null cells compared with full-length TDP1 with amino acid substitutions of an N-terminal serine residue phosphorylated in response to DNA damage. TDP1 forms a stable complex with LigIII170-755, as well as full-length LigIIIα alone or in complex with the DNA repair scaffold protein XRCC1. Small-angle X-ray scattering and negative stain electron microscopy combined with mapping of the interacting regions identified a TDP1/LigIIIα compact dimer of heterodimers in which the two LigIII catalytic cores are positioned in the center, whereas the two TDP1 molecules are located at the edges of the core complex flanked by highly flexible regions that can interact with other repair proteins and SSBs. As TDP1and LigIIIα together repair adducts caused by TOP1 cancer chemotherapy inhibitors, the defined interaction architecture and regulation of this enzyme complex provide insights into a key repair pathway in nonmalignant and cancer cells.

Human histone deacetylase 4 (HDAC4) is a key epigenetic regulator involved in a number of important cellular processes. This makes HDAC4 a promising target for the treatment of several cancers and neurodegenerative diseases, in particular Huntington's disease. HDAC4 is highly regulated by phosphorylation and oxidation, which determine its nuclear or cytosolic localization, and exerts its function through multiple interactions with other proteins, forming multiprotein complexes of varying composition. The catalytic domain of HDAC4 is known to interact with the SMRT/NCOR corepressor complex when the structural zinc-binding domain (sZBD) is intact and forms a closed conformation. Crystal structures of the HDAC4 catalytic domain have been reported showing an open conformation of HDAC4 when bound to certain ligands. Here, we investigated the relevance of this HDAC4 conformation under physiological conditions in solution. We show that proper zinc chelation in the sZBD is essential for enzyme function. Loss of the structural zinc ion not only leads to a massive decrease in enzyme activity, but it also has serious consequences for the overall structural integrity and stability of the protein. However, the Zn 2+ free HDAC4 structure in solution is incompatible with the open conformation. In solution, the open conformation of HDAC4 was also not observed in the presence of a variety of structurally divergent ligands. This suggests that the open conformation of HDAC4 cannot be induced in solution, and therefore cannot be exploited for the development of HDAC4-specific inhibitors., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

DNA methylation is critically involved in the regulation of chromatin states and cell-type-specific gene expression. The exclusive expression of imprinted genes from either the maternal or the paternal allele is regulated by allele-specific DNA methylation at imprinting control regions (ICRs). Aberrant DNA hyper- or hypomethylation at the ICR1 of the H19/IGF2 imprinting locus is characteristic for the imprinting disorders Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS), respectively. In this paper, we performed epigenome editing to induce targeted DNA demethylation at ICR1 in HEK293 cells using dCas9-SunTag and the catalytic domain of TET1. 5-methylcytosine (5mC) levels at the target locus were reduced up to 90% and, 27 days after transient transfection, >60% demethylation was still observed. Consistent with the stable demethylation of CTCF-binding sites within the ICR1, the occupancy of the DNA methylation-sensitive insulator CTCF protein increased by >2-fold throughout the 27 days. Additionally, the H19 expression was increased by 2-fold stably, while IGF2 was repressed though only transiently. Our data illustrate the ability of epigenome editing to implement long-term changes in DNA methylation at imprinting control regions after a single transient treatment, potentially paving the way for therapeutic epigenome editing approaches in the treatment of imprinting disorders.

Peptidoglycan is an essential component of the bacterial cell envelope that contains glycan chains substituted by short peptide stems. Peptide stems are polymerized by D,D-transpeptidases, which make bonds between the amino acid in position four of a donor stem and the third residue of an acceptor stem (4-3 cross-links). Some bacterial peptidoglycans also contain 3-3 cross-links that are formed by another class of enzymes called L,D-transpeptidases which contain a YkuD catalytic domain. In this work, we investigate the formation of unusual bacterial 1-3 peptidoglycan cross-links. We describe a version of the PGFinder software that can identify 1-3 cross-links and report the high-resolution peptidoglycan structure of Gluconobacter oxydans (a model organism within the Acetobacteraceae family). We reveal that G. oxydans peptidoglycan contains peptide stems made of a single alanine as well as several dipeptide stems with unusual amino acids at their C-terminus. Using a bioinformatics approach, we identified a G. oxydans mutant from a transposon library with a drastic reduction in 1-3 cross-links. Through complementation experiments in G. oxydans and recombinant protein production in a heterologous host, we identify an L,D-transpeptidase enzyme with a domain distantly related to the YkuD domain responsible for these non-canonical reactions. This work revisits the enzymatic capabilities of L,D-transpeptidases, a versatile family of enzymes that play a key role in bacterial peptidoglycan remodelling., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract The FokI catalytic domain can be fused to various DNA binding architectures to improve the precision of genome editing tools. However, evaluation of off-target effects is essential for developing these tools. We use Genome-wide Off-target analysis by Two-cell embryo Injection (GOTI) to detect low-frequency off-target editing events in mouse embryos injected with FokI-based architectures. Specifically, we test FokI-heterodimers fused with TALENs, FokI homodimers fused with RYdCas9, or FokI catalytic domains alone resulting in no significant off-target effects. These FokI genome editing systems exhibit undetectable off-target effects in mouse embryos, supporting the further development of these systems for clinical applications.

Mutations in the E3 ubiquitin ligase parkin cause a familial form of Parkinson's disease (PD). Parkin and the mitochondrial kinase PINK1 assure quality control of mitochondria through selective autophagy of mitochondria (mitophagy). Whereas numerous parkin mutations have been functionally and structurally characterized, several PD mutations found in the catalytic Rcat domain of parkin remain poorly understood. Here, we characterize two pathogenic Rcat mutants, T415N and P437L. We demonstrate that both mutants exhibit impaired activity using autoubiquitination and ubiquitin vinyl sulfone assays. We determine the minimal ubiquitin binding segment and show that both mutants display impaired binding of ubiquitin charged on the E2 enzyme. Finally, we use AlphaFold 3 to predict a model of the phospho-parkin:phospho-ubiquitin:ubiquitin-charged E2 complex. The model shows the repressor-element of parkin (REP) and the N-terminal residues of the catalytic domain form a helix to position ubiquitin for transfer from the E2 to parkin. Our results rationalize the pathogenicity of the parkin mutations and deepen our understanding of the active parkin-E2∼Ub complex., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The protozoan parasite Trichomonas vaginalis (Tv) causes trichomoniasis, the most common non-viral sexually transmitted infection in the world. Although Tv has been linked to significant health complications, only two closely related 5-nitroimidazole drugs are approved for its treatment. The emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health, making development of novel anti-Trichomonas compounds an urgent need. The proteasome, a critical enzyme complex found in all eukaryotes has three catalytic subunits, β1, β2, and β5 and has been validated as a drug target to treat trichomoniasis. With the goal of developing tools to study the Tv proteasome, we isolated the enzyme complex and identified inhibitors that preferentially inactivate either one or two of the three catalytic subunits. Using a mass spectrometry-based peptide digestion assay, these inhibitors were used to define the substrate preferences of the β1, β2 and β5 subunits. Subsequently, three model fluorogenic substrates were designed, each specific for one of the catalytic subunits. This novel substrate profiling methodology will allow for individual subunit characterization of other proteasomes of interest. Using the new substrates, we screened a library of 284 peptide epoxyketone inhibitors against Tv and determined the subunits targeted by the most active compounds. The data show that inhibition of the Tv β5 subunit alone is toxic to the parasite. Taken together, the optimized proteasome subunit substrates will be instrumental for understanding the molecular determinants of proteasome specificity and for accelerating drug development against trichomoniasis., (© 2024 The Protein Society.)

Tannins are critical plant defense metabolites, enriched in bark and leaves, that protect against microorganisms and insects by binding to and precipitating proteins. Hydrolyzable tannins contain ester bonds which can be cleaved by tannases-serine hydrolases containing so-called "cap" domains covering their active sites. However, comprehensive insights into the biochemical properties and structural diversity of tannases are limited, especially regarding their cap domains. We here present a code pipeline for structure prediction-based hierarchical clustering to categorize the whole family of bacterial tannases, and have used it to discover new types of cap domains and other structural insertions among these enzymes. Subsequently, we used two recently identified tannases from the gut/soil bacterium Clostridium butyricum as model systems to explore the biochemical and structural properties of the cap domains of tannases. We demonstrate using molecular dynamics and mutagenesis that the cap domain covering the active site plays a major role in enzyme substrate preference, inhibition, and activity-despite not directly interacting with smaller substrates. The present work provides deeper knowledge into the mechanism, structural dynamics, and diversity of tannases. The structure-based clustering approach presents a new way of classifying any other enzyme family, and will be of relevance for enzyme types where activity is influenced by variable loop or insert regions appended to a core protein fold., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

d-Allulose is a low-calorie sweetener with multiple nutritional functions that can be produced through d-fructose isomerization by ketose 3-epimerase (KEase). l-Ribulose 3-epimerase from Arthrobacterglobiformis (AgLRE) is one of the most important enzymes that produce d-allulose; however, its substrate recognition mechanism is unknown. In this study, the crystal structures of AgLRE and its complex with d-allulose and d-fructose were determined. Upon substrate binding, the hydrophobic residues around the active-site entrance move toward the bound substrate. A comparison of AgLRE and other KEase structures revealed that the substrate-binding residues are not the main factors responsible for its marked specificity for d-allulose and d-fructose, but the hydrophobicity of the active site pocket influences substrate recognition. Particularly, the two hydrophobic regions at the active site entrance are the regulatory elements that modulate substrate recognition by AgLRE. This study provides useful information for designing AgLRE to increase its affinity for d-allulose and d-fructose., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Salicylic acid (SA) production in Brassicaceae plants is uniquely accelerated from isochorismate by EPS1, a newly identified enzyme in the BAHD acyltransferase family. We present crystal structures of EPS1 from Arabidopsis thaliana in both its apo and substrate-analog-bound forms. Integrating microsecond-scale molecular dynamics simulations with quantum mechanical cluster modeling, we propose a pericyclic rearrangement lyase mechanism for EPS1. We further reconstitute the isochorismate-derived SA biosynthesis pathway in Saccharomyces cerevisiae, establishing an in vivo platform to examine the impact of active-site residues on EPS1 functionality. Moreover, stable transgenic expression of EPS1 in soybean increases basal SA levels, highlighting the enzyme's potential to enhance defense mechanisms in non-Brassicaceae plants lacking an EPS1 ortholog. Our findings illustrate the evolutionary adaptation of an ancestral enzyme's active site to enable a novel catalytic mechanism that boosts SA production in Brassicaceae plants., Competing Interests: Competing interests: J.K.W. is a member of the Scientific Advisory Board and a shareholder of DoubleRainbow Biosciences, Galixir, and Inari Agriculture, which develop biotechnologies related to natural products, drug discovery, and agriculture. All other authors have no competing interests., (© 2024. The Author(s).)

Ribosome biogenesis is a complex process requiring multiple precursor ribosomal RNA (rRNA) cleavage steps. In archaea, the full set of ribonucleases (RNases) involved in rRNA processing remains to be discovered. A previous study suggested that FAU-1, a conserved protein containing an RNase G/E-like protein domain fused to a domain of unknown function (DUF402), acts as an RNase in archaea. However, the molecular basis of this activity remained so far elusive. Here, we report two X-ray crystallographic structures of RNase G/E-like-DUF402 hybrid proteins from Pyrococcus furiosus and Sulfolobus acidocaldarius, at 2.1 and 2.0 Å, respectively. The structures highlight a structural homology with the 5' RNA recognition domain of Escherichia coli RNase E but no homology with other known catalytic nuclease domains. Surprisingly, we demonstrate that the C-terminal domain of this hybrid protein, annotated as a putative diphosphatase domain, harbors the RNase activity. Our functional analysis also supports a model by which the RNase G/E-like domain acts as a regulatory subunit of the RNase activity. Finally, in vivo experiments in Haloferax volcanii suggest that this RNase participates in the maturation of pre-16S rRNA. Together, our study defines a new RNase family, which we termed the RNase W family, as the first archaea-specific contributor to archaeal ribosome biogenesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The heterotrimeric flavin adenine dinucleotide (FAD) dependent glucose dehydrogenase derived from Burkholderia cepacia (BcGDH) has many exceptional features for its use in glucose sensing-including that this enzyme is capable of direct electron transfer with an electrode in its heterotrimeric configuration. However, this enzyme's high catalytic activity towards not only glucose but also galactose presents an engineering challenge. To increase the substrate specificity of this enzyme, it must be engineered to reduce its activity towards galactose while maintaining its activity towards glucose. To aid in these mutagenesis studies, the crystal structure composed of BcGDH's small subunit and catalytic subunit (BcGDHγα), in complex with D-glucono-1,5-lactone was elucidated and used to construct the three-dimensional model for targeted, site-directed mutagenesis. BcGDHγα was then mutated at three different residues, glycine 322, asparagine 474 and asparagine 475. The single mutations that showed the greatest glucose selectivity were combined to create the resulting mutant, α-G322Q-N474S-N475S. The α-G322Q-N474S-N475S mutant and BcGDHγα wild type were then characterized with dye-mediated dehydrogenase activity assays to determine their kinetic parameters. The α-G322Q-N474S-N475S mutant showed more than a 2-fold increase in V max towards glucose and this mutant showed a lower activity towards galactose in the physiological range (5 mM) of 4.19 U mg -1 , as compared to the wild type, 86.6 U mg -1 . This resulting increase in specificity lead to an 81.7 gal/glc % activity for the wild type while the α-G322Q-N474S-N475S mutant had just 10.9 gal/glc % activity at 5 mM. While the BcGDHγα wild type has high specificity towards galactose, our engineering α-G322Q-N474S-N475S mutant showed concentration dependent response to glucose and was not affected by galactose., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Koji Sode reports financial support was provided by Arkray Inc, Kyoto, Japan. Koji Sode has patent pending to Arkray Inc. Kyoto, Japan. Junko Okuda-Shimazaki has patent pending to Arkray Inc. Katsuhiro Kojima has patent pending to Arkray Inc. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The angiotensin-converting enzyme-2 (ACE2) is a transmembrane glycoprotein, consisting of two segments: a large carboxypeptidase catalytic domain and a small transmembrane collectrin-like segment. This protein plays an essential role in blood pressure regulation, transforming the peptides angiotensin-I and angiotensin-II (vasoconstrictors) into angiotensin-1-9 and angiotensin-1-7 (vasodilators). During the COVID-19 pandemic, ACE2 became best known as the receptor of the S-protein of SARS-CoV-2 coronavirus. The purpose of the following research is to reconstruct the 3D structure of the catalytic domain of the rabbit enzyme rACE2 using its primary amino acid sequence, and then to compare it with the human analog hACE2. For this purpose, we have calculated the electric properties and thermodynamic stability of the two protein globules employing computer programs for protein electrostatics. The analysis of the amino acid content and sequence demonstrates an 85% identity between the two polypeptide chains. The 3D alignment of the catalytic domains of the two enzymes shows coincidence of the α-helix segments, and a small difference in two unstructured segments of the chain. The electric charge of the catalytic domain of rACE2, determined by 70 positively chargeable amino acid residues, 114 negatively chargeable ones, and two positive charges of the Zn 2+ atom in the active center exceeds that of hACE2 by one positively and four negatively chargeable groups; however, in 3D conformation, their isoelectric points pI 5.21 coincide. The surface electrostatic potential is similarly distributed on the surface of the two catalytic globules, but it strongly depends on the pH of the extracellular medium: it is almost positive at pH 5.0 but strongly negative at pH 7.4. The pH dependence of the electrostatic component of the free energy discloses that the 3D structure of the two enzymes is maximally stable at pH 6.5. The high similarity in the 3D structure, as well as in the electrostatic and thermodynamic properties, suggests that rabbit can be successfully used as an animal model to study blood pressure regulation and coronavirus infection, and the results can be extrapolated to humans.

The proline catabolic pathway consisting of proline dehydrogenase (PRODH) and L-glutamate-γ-semialdehyde (GSAL) dehydrogenase (GSALDH) catalyzes the four-electron oxidation of L-proline to L-glutamate. Chemical probes to these enzymes are of interest for their role in cancer and inherited metabolic disease. Here, we report the results of a crystallographic fragment-screening campaign targeting both enzymes. A unique aspect of our approach is the screening of both enzymes simultaneously using crystals of the bifunctional PRODH-GSALDH enzyme, proline utilization A (PutA). A 288-fragment library from Zenobia was screened in crystallo in cocktails of six fragments. Validation X-ray crystallography with individual fragments identified seven crystal hits distributed in the PRODH active site, GSALDH aldehyde substrate-binding site, and GSALDH NAD + adenine-binding site. The fragment bound in the PRODH active site, 4-methoxybenzyl alcohol, is structurally distinct from all known PRODH inhibitors as it lacks an anionic anchor and stabilizes open conformations of the active site, motivating the study of eighteen analogs. In total, thirteen crystal structures with resolutions ranging from 1.32 Å to 1.80 Å were determined, resolving the poses and interactions of seven fragments from the Zenobia library and five analogs of 4-methoxybenzyl alcohol. These results expand the chemical space of probes targeting proline catabolic enzymes and provide new structural information for further inhibitor development.

Apurinic/apyrimidinic endonuclease 1 (APE1) is responsible for the hydrolysis of the phosphodiester bond on the 5' side of an apurinic/apyrimidinic site during base excision repair. Moreover, in DNA, this enzyme can recognize nucleotides containing such damaged bases as 5,6-dihydro-2'-deoxyuridine (DHU), 2'-deoxyuridine (dU), alpha-2'-deoxyadenosine (αA), and 1, N 6-ethenoadenosine (εA). Previously, by pulsed electron-electron double resonance spectroscopy and pre-steady-state kinetic analysis, we have revealed multistep DNA rearrangements during the formation of the catalytic complex. In the present study, the modeling of the eversion trajectory of nucleotides with various damaged bases was performed by directed molecular dynamics simulations. It was found that each damaged base at the beginning of the eversion interacts with protein loop Val196-Arg201, which should be moved to enable further nucleotide eversion. This movement involves a shift in loop Val196-Arg201 away from loop Asn253-Thr257 and requires the disruption of contacts between these loops. The Glu260Ala substitution facilitates the separation of the two loops. Moreover, conformational changes in the Asn253-Thr257 loop should occur in the second half of the lesion eversion trajectory. All these perturbations within the protein globule tend to reduce steric interactions of each damaged base with the protein during the eversion of the nucleotide from DNA and movement to the active site. These perturbations are important determinants of substrate specificity of endonuclease APE1.

RNA oxidation, predominantly through the accumulation of 8-oxo-7,8-dihydroguanosine (8-oxo-rG), represents an important biomarker for cellular oxidative stress. Polynucleotide phosphorylase (PNPase) is a 3'-5' exoribonuclease that has been shown to preferentially recognize 8-oxo-rG-containing RNA and protect E scherichia coli cells from oxidative stress. However, the impact of 8-oxo-rG on PNPase-mediated RNA degradation has not been studied. Here, we show that the presence of 8-oxo-rG in RNA leads to catalytic stalling of E. coli PNPase through in vitro RNA degradation experiments and electrophoretic analysis. We also link this stalling to the active site of the enzyme through resolution of single-particle cryo-EM structures for PNPase in complex with singly or doubly oxidized RNA oligonucleotides. Following identification of Arg399 as a key residue in recognition of both single and sequential 8-oxo-rG nucleotides, we perform follow-up in vitro analysis to confirm the importance of this residue in 8-oxo-rG-specific PNPase stalling. Finally, we investigate the effects of mutations to active site residues implicated in 8-oxo-rG binding through E. coli cell growth experiments under H 2 O 2 -induced oxidative stress. Specifically, Arg399 mutations show significant effects on cell growth under oxidative stress. Overall, we demonstrate that 8-oxo-rG-specific stalling of PNPase is relevant to bacterial survival under oxidative stress and speculate that this enzyme might associate with other cellular factors to mediate this stress., Competing Interests: Competing interests statement:The authors declare no competing interest.

In recent years, a convenient phosphatase-coupled sulfotransferase assay method has been proven to be applicable to most sulfotransferases. The central principle of the method is that phosphatase specifically degrades 3'-phosphoadenosine-5'-phosphate (PAP) and leaves 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Our group previously acquired a yeast 3',5'-bisphosphate nucleotidase (YND), which showed a higher catalytic activity for PAP than PAPS and could be a potential phosphatase for the sulfotransferase assay. Here, we obtained a beneficial mutant of YND with markedly improved substrate specificity towards PAP via rational design. Of 9 chosen mutation sites in the active site pocket, the mutation G236D showed the best specificity for PAP. After optimization of the reaction conditions, the mutant YND G236D displayed a 4.8-fold increase in the catalytic ratio PAP/PAPS compared to the wild-type. We subsequently applied YND G236D to the assay of human SULT1A1 and SULT1A3 with their known substrate 1-naphthol, indicating that the mutant could be used to evaluate sulfotransferase activity by colorimetry. Analysis of the MD simulation results revealed that the improved substrate specificity of the mutant towards PAP may stem from a more stable protein conformation and the changed flexibility of key residues in the entrance of the substrate tunnel. This research will provide a valuable reference for the development of efficient sulfotransferase activity assays.

Subtilisin-like proteins are serine proteases that use two types of catalytic triads: Ser-His-Asp and Ser-Glu-Asp. Here, we investigate the two known families of subtilisin-like proteins, the subtilases (Ser-His-Asp triad) and the serine-carboxyl proteinases (Ser-Glu-Asp triad), and describe the local structural arrangements (cores) that govern the catalytic residues in these proteins. We show the separation of the cores into conserved structural zones, which can be repeatedly found in different structures, and compare the structural cores in subtilisin-like proteins with those in trypsin-like serine proteases and alpha/beta-hydrolases.

ACMSD (α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase) is a key metalloenzyme critical for regulating de novo endogenous NAD + /NADH biosynthesis through the tryptophan-kynurenine pathway. This decarboxylase is a recognized target implicated in mitochondrial diseases and neurodegenerative disorders. However, unraveling its enzyme-substrate complex has been challenging due to its high catalytic efficiency. Here, we present a combined biochemical and structural study wherein we determined the crystal structure of ACMSD in complex with malonate. Our analysis revealed significant rearrangements in the active site, particularly in residues crucial for ACMS decarboxylation, including Arg51, Arg239∗ (a residue from an adjacent subunit), His228, and Trp194. Docking modeling studies proposed a putative ACMS binding mode. Additionally, we found that ACMSD catalyzes oxaloacetic acid (OAA) tautomerization at a rate of 6.51 ± 0.42 s -1 but not decarboxylation. The isomerase activity of ACMSD on OAA warrants further investigation in future biological studies. Subsequent mutagenesis studies and crystallographic analysis of the W194A variant shed light on the roles of specific second-coordination sphere residues. Our findings indicate that Arg51 and Arg239∗ are crucial for OAA tautomerization. Moreover, our comparative analysis with related isomerase superfamily members underscores a general strategy employing arginine residues to promote OAA isomerization. Given the observed isomerase activity of ACMSD on OAA and its structural similarity to ACMS, we propose that ACMSD may facilitate isomerization to ensure ACMS is in the optimal tautomeric form for subsequent decarboxylation initiated by the zinc-bound hydroxide ion. Overall, these findings deepen the understanding of the structure and function of ACMSD, offering insights into potential therapeutic interventions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The methyltransferase ribozyme SMRZ-1 utilizes S-adenosyl-methionine (SAM) and Cu (II) ions to methylate RNA. A comparison of the SAM-bound and unbound RNA structures has shown a conformational change in the RNA. However, the contribution of specific interactions and the role of a pseudo-triplex motif in the catalytic center on the methylation reaction is not completely understood. In this study, we have used atomic substitutions and mutational analysis to investigate the reaction specificity and the key interactions required for catalysis. Substitution of the fluorescent nucleotide 2-aminopurine within the active ribozyme enabled the conformational dynamics of the RNA upon co-factor binding to be explored using fluorescence spectroscopy. We show that fast co-factor binding (t 1/2 ∼ 0.7 s) drives a conformational change in the RNA to facilitate methyl group transfer. The importance of stacking interactions at the pseudo-triplex motif and chelation of the Cu (II) ion were shown to be essential for SAM binding., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The search for novel synthetic tools to prepare industrial chemicals in a safer and greener manner is a continuing challenge in synthetic chemistry. In this manuscript, we report the discovery, characterization, and synthetic potential of two novel aryl-alcohol oxidases from bacteria which are able to oxidize a variety of aliphatic and aromatic alcohols with efficiencies up to 4970 min -1  mM -1 . Both enzymes have shown a reasonable thermostability (thermal melting temperature values of 50.9 and 48.6 °C for ShAAO and SdAAO, respectively). Crystal structures revealed an unusual wide-open entrance to the active-site pockets compared to that previously described for traditional fungal aryl-alcohol oxidases, which could be associated with differences observed in substrate scope, catalytic efficiency, and other functional properties. Preparative-scale reactions and the ability to operate at high substrate loadings also demonstrate the potential of these enzymes in synthetic chemistry with total turnover numbers > 38000. Moreover, their availability as soluble and active recombinant proteins enabled their use as cell-free extracts which further highlights their potential for the large-scale production of carbonyl compounds. KEY POINTS: • Identification and characterization of two novel bacterial aryl-alcohol oxidases • Crystal structures reveal wide-open active-site pockets, impacting substrate scope • Total turnover numbers and cell-free extracts demonstrate the synthetic potential., (© 2024. The Author(s).)

It is broadly recognized that intramolecular electric fields, produced by the protein scaffold and acting on the active site, facilitate enzymatic catalysis. This field effect can be described by several theoretical models, each of which is intuitive to varying degrees. In this contribution, we show that a fundamental effect of electric fields is to generate electrostatic potentials that facilitate the energetic alignment of reactant frontier orbitals. We apply this model to demystify the impact of electric fields on high-valent iron-oxo heme proteins: catalases, peroxidases, and peroxygenases/monooxygenases. Specifically, we show that this model easily accounts for the observed field-induced changes to the spin distribution within peroxidase active sites and explains the transition between epoxidation and hydroxylation pathways seen in Cytochrome P450 active site models. Thus, for the intuitive interpretation of the chemical effect of the field, the strategy involves analyzing the response of the orbitals of active site fragments, and their energetic alignment. We note that the energy difference between fragment orbitals involved in charge redistribution acts as a measure for the chemical hardness/softness of the reactive complex. This measure, and its sensitivity to electric fields, offers a single parameter model from which to quantitatively assess the effects of electric fields on reactivity and selectivity. Thus, the model provides an additional perspective to describe electrostatic preorganization and offers ways for its manipulation., Competing Interests: Competing interests statement:The authors declare no competing interest.

Iodothyronine deiodinases (Dio) are selenocysteine-containing membrane enzymes that activate and inactivate the thyroid hormones (TH) through reductive iodide eliminations. The three deiodinase isoforms are homodimers sharing highly conserved amino acid sequences, but they differ in their regioselectivities for the deiodination reaction and regulatory features. We have now solved a crystal structure of the mouse deiodinase 2 (Dio2) catalytic domain. It reveals a high overall similarity to the deiodinase 3 structure, supporting the proposed common mechanism, but also Dio2-specific features, likely mediating its unique properties. Activity studies with an artificially enforced Dio dimer further confirm that dimerization is required for activity and requires both the catalytic core and the enzyme's N-terminus. Cross-linking studies reveal the catalytic core's dimerization interface, providing insights into the architecture of the complete, active Dio homodimer.

Mutations in the parkin gene product Parkin give rise to autosomal recessive juvenile parkinsonism. Parkin is an E3 ubiquitin ligase that is a critical participant in the process of mitophagy. Parkin has a complex structure that integrates several allosteric signals to maintain precise control of its catalytic activity. Though its allosterically controlled structural reorganization has been extensively characterized by crystallography, the energetics and mechanisms of allosteric regulation of Parkin are much less well understood. Allostery is fundamentally linked to the energetics of the cooperative (sub)structure of the protein. Herein, we examine the mechanism of allosteric activation by phosphorylated ubiquitin binding to the enzymatic core of Parkin, which lacks the antagonistic Ubl domain. In this way, the allosteric effects of the agonist phosphorylated ubiquitin can be isolated. Using native-state hydrogen exchange monitored by mass spectrometry, we find that the five structural domains of the core of Parkin are energetically distinct. Nevertheless, association of phosphorylated ubiquitin destabilizes structural elements that bind the ubiquitin-like domain antagonist while promoting the dissociation of the catalytic domain and energetically poises the protein for transition to the fully activated structure.

S2P intramembrane metalloproteases regulate diverse signaling pathways across all three domains of life. However, the mechanism by which S2P metalloproteases engage substrates and catalyze peptide hydrolysis within lipid membranes has remained elusive. Here we determine the cryo-EM structure of the S2P family intramembrane metalloprotease SpoIVFB from Bacillus subtilis bound to its native substrate Pro-σ K . The structure and accompanying biochemical data demonstrate that SpoIVFB positions Pro-σ K at the enzyme active site through a β-sheet augmentation mechanism, and reveal key interactions between Pro-σ K and the interdomain linker connecting SpoIVFB transmembrane and CBS domains. The cryo-EM structure and molecular dynamics simulation reveal a plausible path for water to access the membrane-buried active site of SpoIVFB, and suggest a possible role of membrane lipids in facilitating substrate capture. These results provide key insight into how S2P intramembrane metalloproteases capture and position substrates for hydrolytic proteolysis within the hydrophobic interior of a lipid membrane., (© 2024. The Author(s).)

ATP-grasp superfamily enzymes contain a hand-like ATP-binding fold and catalyze a variety of reactions using a similar catalytic mechanism. More than 30 protein families are categorized in this superfamily, and they are involved in a plethora of cellular processes and human diseases. Here, we identify C12orf29 (RLIG1) as an atypical ATP-grasp enzyme that ligates RNA. Human RLIG1 and its homologs autoadenylate on an active site Lys residue as part of a reaction intermediate that specifically ligates RNA halves containing a 5'-phosphate and a 3'-hydroxyl. RLIG1 binds tRNA in cells and can ligate tRNA within the anticodon loop in vitro. Transcriptomic analyses of Rlig1 knockout mice revealed significant alterations in global tRNA levels in the brains of female mice, but not in those of male mice. Furthermore, crystal structures of a RLIG1 homolog from Yasminevirus bound to nucleotides revealed a minimal and atypical RNA ligase fold with a conserved active site architecture that participates in catalysis. Collectively, our results identify RLIG1 as an RNA ligase and suggest its involvement in tRNA biology., Competing Interests: Competing interests statement:The authors declare no competing interest.

S-omeprazole and R-rabeprazole are important proton pump inhibitors (PPIs) used for treating peptic disorders. They can be biosynthesized from the corresponding sulfide catalyzed by Baeyer-Villiger monooxygenases (BVMOs). During the development of BVMOs for target sulfoxide preparation, stereoselectivity and overoxidation degree are important factors considered most. In the present study, LnPAMO-Mu15 designed previously and TtPAMO from Thermothelomyces thermophilus showed high (S)- and (R)-configuration stereoselectivity respectively towards thioethers. TtPAMO was found to be capable of oxidating omeprazole sulfide (OPS) and rabeprazole sulfide (RPS) into R-omeprazole and R-rabeprazole respectively. However, the overoxidation issue existed and limited the application of TtPAMO in the biosynthesis of sulfoxides. The structural mechanisms for adverse stereoselectivity between LnPAMO-Mu15 and TtPAMO towards OPS and the overoxidation of OPS by TtPAMO were revealed, based on which, TtPAMO was rationally designed focused on the flexibility of loops near catalytic sites. The variant TtPAMO-S482Y was screened out with lowest overoxidation degree towards OPS and RPS due to the decreased flexibility of catalytic center than TtPAMO. The success in this study not only proved the rationality of the overoxidation mechanism proposed in this study but also provided hints for the development of BVMOs towards thioether substrate for corresponding sulfoxide preparation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bacillus circulans xylanase (BcX) from the glycoside hydrolase family 11 degrades xylan through a retaining, double-displacement mechanism. The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large, active site cleft, with six subsites allowing the binding of six xylose units. Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways. In the crystal structure of the catalytically inactive variant BcX E78Q, the substrate xylotriose is observed in the active site, as well as bound to the known secondary binding site and a third site on the protein surface. Nuclear magnetic resonance (NMR) titrations with xylose oligomers of different lengths yield nonlinear chemical shift trajectories for active site nuclei resonances, indicative of multiple binding orientations for these substrates for which binding and dissociation are in fast exchange on the NMR timescale, exchanging on the micro- to millisecond timescale. Active site binding can be modeled with a 2 : 1 model with dissociation constants in the low and high millimolar range. Extensive mutagenesis of active site residues indicates that tight binding occurs in the glycon binding site and is stabilized by Trp9 and the thumb region. Mutations F125A and W71A lead to large structural rearrangements. Binding at the glycon site is sensed throughout the active site, whereas the weak binding mostly affects the aglycon site. The interactions with the two active site locations are largely independent of each other and of binding at the secondary binding site., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated Pseudomonas exotoxin (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in Escherichia coli , achieving high solubility and purity post-purification. In vitro cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC 50 values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.

Regulatory adenine nucleotide-binding cystathionine β-synthase (CBS) domains are widespread in proteins; however, information on the mechanism of their modulating effects on protein function is scarce. The difficulty in obtaining structural data for such proteins is ascribed to their unusual flexibility and propensity to form higher-order oligomeric structures. In this study, we deleted the most movable domain from the catalytic part of a CBS domain-containing bacterial inorganic pyrophosphatase (CBS-PPase) and characterized the deletion variant both structurally and functionally. The truncated CBS-PPase was inactive but retained the homotetrameric structure of the full-size enzyme and its ability to bind a fluorescent AMP analog (inhibitor) and diadenosine tetraphosphate (activator) with the same or greater affinity. The deletion stabilized the protein structure against thermal unfolding, suggesting that the deleted domain destabilizes the structure in the full-size protein. A "linear" 3D structure with an unusual type of domain swapping predicted for the truncated CBS-PPase by Alphafold2 was confirmed by single-particle electron microscopy. The results suggest a dual role for the CBS domains in CBS-PPase regulation: they allow for enzyme tetramerization, which impedes the motion of one catalytic domain, and bind adenine nucleotides to mitigate or aggravate this effect.