Rossmann Fold Research Articles

Structural Basis of Protein Arginine Methyltransferase Activation by a Catalytically Dead Homolog (Prozyme)

Prozymes are pseudoenzymes that stimulate the function of weakly active enzymes through complex formation. The major Trypanosoma brucei protein arginine methyltransferase, TbPRMT1 enzyme (ENZ), requires TbPRMT1 prozyme (PRO) to form an active heterotetrameric complex. Here, we present the X-ray crystal structure of the TbPRMT1 ENZ–Δ52PRO tetrameric complex with the cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.4 Å resolution. The individual ENZ and PRO units adopt the highly-conserved PRMT domain architecture and form an antiparallel heterodimer that corresponds to the canonical homodimer observed in all previously reported PRMTs. In turn, two such heterodimers assemble into a tetramer both in the crystal and in solution with twofold rotational symmetry. ENZ is unstable in absence of PRO and incapable of forming a homodimer due to a steric clash of an ENZ-specific tyrosine within the dimerization arm, rationalizing why PRO is required to complement ENZ to form a PRMT dimer that is necessary, but not sufficient for PRMT activity. The PRO structure deviates from other, active PRMTs in that it lacks the conserved η2 310-helix within the Rossmann fold, abolishing cofactor binding. In addition to its chaperone function for ENZ, PRO substantially contributes to substrate binding. Heterotetramerization is required for catalysis, as heterodimeric ENZ–PRO mutants lack binding affinity and methyltransferase activity toward the substrate protein TbRGG1. Together, we provide a structural basis for TbPRMT1 ENZ activation by PRO heterotetramer formation, which is conserved across all kinetoplastids, and describe a chaperone function of the TbPRMT1 prozyme, which represents a novel mode of PRMT regulation.

The modular structure of α/β-hydrolases.

The α/β-hydrolase fold family is highly diverse in sequence, structure and biochemical function. To investigate the sequence-structure-function relationships, the Lipase Engineering Database (https://led.biocatnet.de) was updated. Overall, 280638 protein sequences and 1557 protein structures were analysed. All α/β-hydrolases consist of the catalytically active core domain, but they might also contain additional structural modules, resulting in 12 different architectures: core domain only, additional lids at three different positions, three different caps, additional N- or C-terminal domains and combinations of N- and C-terminal domains with caps and lids respectively. In addition, the α/β-hydrolases were distinguished by their oxyanion hole signature (GX-, GGGX- and Y-types). The N-terminal domains show two different folds, the Rossmann fold or the β-propeller fold. The C-terminal domains show a β-sandwich fold. The N-terminal β-propeller domain and the C-terminal β-sandwich domain are structurally similar to carbohydrate-binding proteins such as lectins. The classification was applied to the newly discovered polyethylene terephthalate (PET)-degrading PETases and MHETases, which are core domain α/β-hydrolases of the GX- and the GGGX-type respectively. To investigate evolutionary relationships, sequence networks were analysed. The degree distribution followed a power law with a scaling exponent γ=1.4, indicating a highly inhomogeneous network which consists of a few hubs and a large number of less connected sequences. The hub sequences have many functional neighbours and therefore are expected to be robust toward possible deleterious effects of mutations. The cluster size distribution followed a power law with an extrapolated scaling exponent τ=2.6, which strongly supports the connectedness of the sequence space of α/β-hydrolases. DATABASE: Supporting data about domains from other proteins with structural similarity to the N- or C-terminal domains of α/β-hydrolases are available in Data Repository of the University of Stuttgart (DaRUS) under doi: https://doi.org/10.18419/darus-458.

The crystal structure of sulfiredoxin from Arabidopsis thaliana revealed a more robust antioxidant mechanism in plants

Typical 2-cysteine peroxiredoxins (2-Cys Prxs) are critical peroxidase sensors and could be deactivated by the hyperoxidation under oxidative stress. In plants, 2-Cys Prxs present at a high level in chloroplasts and are repaired by Sulfiredoxin. Whereas many studies have explored the mechanism of Sulfiredoxin from Homo sapiens (HsSrx), the molecular mechanism of Sulfiredoxin in plants with unique photosynthesis remains unclear. Here we report the crystal structure of Sulfiredoxin from Arabidopsis thaliana (AtSrx), which displayed a typical ParB/Srx fold with an ATP bound at a conservative nucleotide binding motif GCHR. Both the ADP binding pocket and the putative AtSrx-AtPrxA interaction surface of AtSrx are more positively charged comparing to HsSrx, suggesting a robust mechanism of AtSrx. These features illustrate the unique mechanisms of AtSrx, which are vital for figure out the strategies of plants to cope with oxidation stress.

The mitochondrial intermembrane space-facing proteins Mcp2 and Tgl2 are involved in yeast lipid metabolism.

Mitochondria are unique organelles harboring two distinct membranes, the mitochondrial inner and outer membrane (MIM and MOM, respectively). Mitochondria comprise only a subset of metabolic pathways for the synthesis of membrane lipids; therefore most lipid species and their precursors have to be imported from other cellular compartments. One such import process is mediated by the ER mitochondria encounter structure (ERMES) complex. Both mitochondrial membranes surround the hydrophilic intermembrane space (IMS). Therefore, additional systems are required that shuttle lipids between the MIM and MOM. Recently, we identified the IMS protein Mcp2 as a high-copy suppressor for cells that lack a functional ERMES complex. To understand better how mitochondria facilitate transport and biogenesis of lipids, we searched for genetic interactions of this suppressor. We found that MCP2 has a negative genetic interaction with the gene TGL2 encoding a neutral lipid hydrolase. We show that this lipase is located in the intermembrane space of the mitochondrion and is imported via the Mia40 disulfide relay system. Furthermore, we show a positive genetic interaction of double deletion of MCP2 and PSD1, the gene encoding the enzyme that synthesizes the major amount of cellular phosphatidylethanolamine. Finally, we demonstrate that the nucleotide-binding motifs of the predicted atypical kinase Mcp2 are required for its proper function. Taken together, our data suggest that Mcp2 is involved in mitochondrial lipid metabolism and an increase of this involvement by overexpression suppresses loss of ERMES.

Functional prediction, characterization, and categorization of operome from Acetoanaerobium sticklandii DSM 519

Acetoanaerobium sticklandii DSM 519 is a hyper-ammonia producing anaerobic bacterium that can be able utilizes amino acids as sole carbon and energy sources for its growth and energetic metabolism. A lack of knowledge on its molecular machinery and 30.5% conserved hypothetical proteins (HPs; operome) hinders the successful utility in biofuel applications. In this study, we have predicted, characterized and categorized its operome whose functions are still not determined accurately using a combined bioinformatics approach. The functions of 64 of the 359 predicted HPs are involved in diverse metabolic subsystems. A. sticklandii operome has consisted of 16% Rossmann fold and 46% miscellaneous folds. Subsystems-based technology has classified 51 HPs contributing to the small-molecular reactions, 26 in macromolecular reactions and 12 in the biosynthesis of cofactors, prosthetic groups and electron carriers. A generality of functions predicted from its operome contributed to the cell cycle, amino acid metabolism, membrane transport, and regulatory processes. Many of them have duplicated functions as paralogs in this genome. A. sticklandii has the ability to compete with invading microorganisms and tolerate abiotic stresses, which can be overwhelmed by the predicted functions of its operome. Results of this study revealed that it has specialized systems for amino acid catabolism-directed solventogenesis and acidogenesis but the level of gene expression may determine the metabolic function in amino acid fermenting niches in the rumina of cattle. As shown by our analysis, the predicted functions of its operome allow us for a better understanding of the growth and physiology at systems-scale.

Catalyst: Can Systems Chemistry Unravel the Mysteries of the Chemical Origins of Life?

Daniela Kroiss obtained her MSc degree in biotechnology at BOKU and is currently a PhD student in the biochemistry program at CUNY, where she works on a joint project between the Nanoscience Initiative and the University of Strathclyde. Gonen Ashkenasy is head of the Laboratory for Systems Chemistry at the Ben-Gurion University Chemistry Department. His research focuses on the design and analysis of synthetic replication networks. Adam Braunschweig earned his PhD in organic chemistry at the University of California and is an associate professor in the CUNY Nanoscience Initiative. His research focuses on the interface of organic chemistry with biology and material science. Tell Tuttle obtained his PhD in theoretical chemistry at Goteborg University and is a professor of theoretical chemistry at the University of Strathclyde. His research focuses on the directed discovery of peptide-based materials. Rein Ulijn gained his PhD in physical chemistry at the University of Strathclyde and is founding director of the Nanoscience Initiative at the CUNY Advanced Science Research Center. His research focuses on nanotechnology inspired by living systems.

Combing Transcriptomes for Secrets of Deep-Sea Survival: Environmental Diversity Drives Patterns of Protein Evolution.

Ctenophores, also known as comb jellies, live across extremely broad ranges of temperature and hydrostatic pressure in the ocean. Because various ctenophore lineages adapted independently to similar environmental conditions, Phylum Ctenophora is an ideal system for the study of protein adaptation to extreme environments in a comparative framework. We present such a study here, using a phylogenetically-informed method to compare sequences of four essential metabolic enzymes across gradients of habitat depth and temperature. This method predicts convergent adaptation to these environmental parameters at the amino acid level, providing a novel view of protein adaptation to extreme environments and demonstrating the power and relevance of phylogenetic comparison applied to multi-species transcriptomic datasets from early-diverging metazoa. Across all four enzymes analyzed, 46 amino acid sites were associated with depth-adaptation, 59 with temperature-adaptation, and 56 with both. Sites predicted to be depth- and temperature-adaptive occurred consistently near Rossmann fold cofactor binding motifs and disproportionately in solvent-exposed regions of the protein. These results suggest that the hydrophobic effect and ligand binding may mediate efficient enzyme function at different hydrostatic pressures and temperatures. Using predicted adaptive site maps, such mechanistic hypotheses can now be tested via mutagenesis.

DprA Is Essential for Natural Competence in Riemerella anatipestifer and Has a Conserved Evolutionary Mechanism.

Riemerella anatipestifer ATCC11845 (RA ATCC11845) is naturally competent. However, the genes involved in natural transformation in this species remain largely unknown. Bioinformatic analysis predicts that DprA of RA (DprARa) has three domains: a sterile alpha motif (SAM), a Rossmann fold (RF) domain and a Z-DNA-binding domain (Zα). Inactivation of dprA abrogated natural transformation in RA ATCC11845, and this effect was restored by the expression of dprA in trans. The dprA with SAM and RF domains of Streptococcus pneumoniae and the dprA with RF and Zα domains of Helicobacter pylori was able to restore natural transformation in the RA ATCC11845 dprA mutant. An Arg123 mutation in the RF domain of R. anatipestifer was not able to restore natural transformation of the RA ATCC11845 dprA mutant. Furthermore, DprAR123E abolished its ability to bind DNA, suggesting that the RF domain is essential for the function of DprA. Finally, the dprA of Fusobacterium naviforme which has not been reported to be natural competent currently was partially able to restore natural transformation in RA ATCC11845 dprA mutant. These results collectively suggest that DprA has a conserved evolutionary mechanism.

A Type III CRISPR Ancillary Ribonuclease Degrades Its Cyclic Oligoadenylate Activator

Cyclic oligoadenylate (cOA) secondary messengers are generated by type III CRISPR systems in response to viral infection. cOA allosterically activates the CRISPR ancillary ribonucleases Csx1/Csm6, which degrade RNA non-specifically using a HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) active site. This provides effective immunity but can also lead to growth arrest in infected cells, necessitating a means to deactivate the ribonuclease once viral infection has been cleared. In the crenarchaea, dedicated ring nucleases degrade cA4 (cOA consisting of 4 AMP units), but the equivalent enzyme has not been identified in bacteria. We demonstrate that, in Thermus thermophilus HB8, the uncharacterized protein TTHB144 is a cA4-activated HEPN ribonuclease that also degrades its activator. TTHB144 binds and degrades cA4 at an N-terminal CARF (CRISPR-associated Rossman fold) domain. The two activities can be separated by site-directed mutagenesis. TTHB144 is thus the first example of a self-limiting CRISPR ribonuclease.

Remarkable diversification of bacterial azoreductases: primary sequences, structures, substrates, physiological roles, and biotechnological applications.

Azoreductases reductively cleave azo linkages by using NAD(P)H as an electron donor. The enzymes are widely found in bacteria and act on numerous azo dyes, which allow various unique applications. This review describes primary amino acid sequences, structures, substrates, physiological roles, and biotechnological applications of bacterial azoreductases to discuss their remarkable diversification. According to primary sequences, azoreductases were classified phylogenetically into four main clades. Most members of clades I-III are flavoproteins, whereas clade IV members include flavin-free azoreductases. Clades I and II prefer NADPH and NADH, respectively, as electron donors, whereas other members generally use both. Several enzymes formed no clades; moreover, some bacteria produce azoreductases with longer primary structures than those hitherto identified, which implies further diversification of bacterial azoreductases. The crystal structures commonly reveal the Rossmann folds; however, ternary structures are moderately varied with different quaternary conformation. Although physiological roles are obscure, several azoreductases have been shown to act on metabolites such as flavins, quinones, and metal ions more efficiently than on azo dyes. Considering that many homologs exclusively act on these metabolites, it is possible that azoreductases are actually side activities of versatile reductases that act on various substrates with different specificities. In parallel, this idea raises the possibility that homologous enzymes, even if these are already defined as other types of reductases, widely harbor azoreductase activities. Although azoreductases for which their genes have been identified are not abundant, it may be simple to identify azoreductases of biotechnological importance that have novel substrate specificities.

A CRISPR-associated Rossmann fold (CARF) domain regulates transcription of an RNA repair system in Escherichia coli

CRISPR-associated Rossmann fold (CARF) domain signalling underpins the modulation of CRISPR-Cas systems. Unlike the majority of known CARF domains associated with nuclease activity in CRISPR-Cas systems, the CARF domain of the transcriptional regulator RtcR modulates the opposite function by activating an RNA end sealing system. The Rtc RNA repair system in Escherichia coli consists ofthe universally conserved RNA cyclase RtcA and RNA ligase RtcB, and is known to be induced by antibiotics and oxidative stress. We aim to investigate the CARF domain mediated transcriptional regulation of the Rtc system in vivo and in vitro. A reporter based assay confirmed that the RtcR CARF domain has a negative regulatory effect on RtcR activity and subsequent induction of the rtcBA operon. Both predictive modelling and site-directed mutagenesis suggest the Rtc induction is not due to oxidation of cysteines present in the RtcR regulatory CARF domain. Interestingly, the enzymes RtcA and RtcB, products of the actions of RtcR as a transcription regulator, are required for RtcR to stimulate expression of the rtcBA operon and directly bind to the RtcR CARF domain as shown in vivo by two-hybrid and in vitro by gel filtration. Given that CARF domains are known to be activated by RNA binding, a range of RNA molecules linked to the Rtc system or CARF domains in general are currently being investigated as potential inducers in an in vitro transcription system. Together, our data indicate an expanded range for CARF domain signalling, including via protein-protein interactions.

Structure of glycerol dehydrogenase (GldA) from Escherichia coli.

Escherichia coli (strain K-12, substrain MG1655) glycerol dehydrogenase (GldA) is required to catalyze the first step in fermentative glycerol metabolism. The protein was expressed and purified to homogeneity using a simple combination of heat-shock and chromatographic methods. The high yield of the protein (∼250 mg per litre of culture) allows large-scale production for potential industrial applications. Purified GldA exhibited a homogeneous tetrameric state (∼161 kDa) in solution and relatively high thermostability (Tm = 65.6°C). Sitting-drop sparse-matrix screens were used for protein crystallization. An optimized condition with ammonium sulfate (2 M) provided crystals suitable for diffraction, and a binary structure containing glycerol in the active site was solved at 2.8 Å resolution. Each GldA monomer consists of nine β-strands, thirteen α-helices, two 310-helices and several loops organized into two domains, the N- and C-terminal domains; the active site is located in a deep cleft between the two domains. The N-terminal domain contains a classic Rossmann fold for NAD+ binding. The O1 and O2 atoms of glycerol serve as ligands for the tetrahedrally coordinated Zn2+ ion. The orientation of the glycerol within the active site is mainly stabilized by van der Waals and electrostatic interactions with the benzyl ring of Phe245. Computer modeling suggests that the glycerol molecule is sandwiched by the Zn2+ and NAD+ ions. Based on this, the mechanism for the relaxed substrate specificity of this enzyme is also discussed.

Elucidation of the mechanism of disulfide exchange between staphylococcal thioredoxin2 and thioredoxin reductase2: A structural insight

The redox homeostasis of cytoplasm is maintained by a series of disulfide exchange reactions mediated by proteins belonging to the thioredoxin superfamily. Thioredoxin and thioredoxin reductase, being the major members of the family, play a key role in oxidative stress response of Staphylococcus aureus. In this report, we have identified and characterised an active thioredoxin system of the mentioned pathogen. Crystal structure of thioredoxin2 (SaTrx2) in its reduced form reveals that it contains the conserved redox active WCXXC motif and a thioredoxin fold. Thioredoxin reductase2 (SaTR2) is a flavoprotein and consists of two Rossmann folds as the binding sites for FAD and NADPH. Crystal structure of the SaTR2 holoenzyme shows that the protein consists of two domains and the catalytic site comprises of an intramolecular disulfide bond formed between two sequentially distal cysteine residues. Biophysical and biochemical studies unveil that SaTrx2 and SaTR2 can physically interact in solution and in the course of sustaining the redox equilibrium, the latter reduces the former. Molecular docking has been performed to illustrate the interface formed between SaTrx2 and SaTR2 during the disulfide exchange reaction.

N-glycan Utilization by Bifidobacterium Gut Symbionts Involves a Specialist β-Mannosidase

Bifidobacteria represent one of the first colonizers of human gut microbiota, providing to this ecosystem better health and nutrition. To maintain a mutualistic relationship, they have enzymes to degrade and use complex carbohydrates non-digestible by their hosts. To succeed in the densely populated gut environment, they evolved molecular strategies that remain poorly understood. Herein, we report a novel mechanism found in probiotic Bifidobacteria for the depolymerization of the ubiquitous 2-acetamido-2-deoxy-4-O-(β-d-mannopyranosyl)-d-glucopyranose (Man-β-1,4-GlcNAc), a disaccharide that composes the universal core of eukaryotic N-glycans. In contrast to Bacteroidetes, these Bifidobacteria have a specialist and strain-specific β-mannosidase that contains three distinctive structural elements conferring high selectivity for Man-β-1,4-GlcNAc: a lid that undergoes conformational changes upon substrate binding, a tryptophan residue swapped between the two dimeric subunits to accommodate the GlcNAc moiety, and a Rossmann fold subdomain strategically located near to the active site pocket. These key structural elements for Man-β-1,4-GlcNAc specificity are highly conserved in Bifidobacterium species adapted to the gut of a wide range of social animals, including bee, pig, rabbit, and human. Together, our findings uncover an unprecedented molecular strategy employed by Bifidobacteria to selectively uptake carbohydrates from N-glycans in social hosts.

Deciphering Determinants in Ribosomal Methyltransferases That Confer Antimicrobial Resistance.

Post-translational methylation of rRNA at select positions is a prevalent resistance mechanism adopted by pathogens. In this work, KsgA, a housekeeping ribosomal methyltransferase (rMtase) involved in ribosome biogenesis, was exploited as a model system to delineate the specific targeting determinants that impart substrate specificity to rMtases. With a combination of evolutionary and structure-guided approaches, a set of chimeras were created that altered the targeting specificity of KsgA such that it acted similarly to erythromycin-resistant methyltransferases (Erms), rMtases found in multidrug-resistant pathogens. The results revealed that specific loop embellishments on the basic Rossmann fold are key determinants in the selection of the cognate RNA. Moreover, in vivo studies confirmed that chimeric constructs are competent in imparting macrolide resistance. This work explores the factors that govern the emergence of resistance and paves the way for the design of specific inhibitors useful in reversing antibiotic resistance.

55 Years of the Rossmann Fold.

The Rossmann fold is one of the most commonly observed structural domains in proteins. The fold is composed of consecutive alternating β-strands and α-helices that form a layer of β-sheet with one (or two) layer(s) of α-helices. Here, we will discuss the Rossmann fold starting from its discovery 55years ago, then overview entries of the fold in the major protein classification databases, SCOP and CATH, as well as the number of the occurrences of the fold in genomes. We also discuss the Rossmann fold as an interesting target of protein engineering as the site-directed mutagenesis of the fold can alter the ligand-binding specificity of the structure.

Crystal Structure of Aldehyde Dehydrogenase 16 Reveals Trans-Hierarchical Structural Similarity and a New Dimer

The aldehyde dehydrogenase (ALDH) superfamily is a vast group of enzymes that catalyze the NAD+-dependent oxidation of aldehydes to carboxylic acids. ALDH16 is perhaps the most enigmatic member of the superfamily, owing to its extra C-terminal domain of unknown function and the absence of the essential catalytic cysteine residue in certain non-bacterial ALDH16 sequences. Herein we report the first production of recombinant ALDH16, the first biochemical characterization of ALDH16, and the first crystal structure of ALDH16. Recombinant expression systems were generated for the bacterial ALDH16 from Loktanella sp. and human ALDH16A1. Four high-resolution crystal structures of Loktanella ALDH16 were determined. Loktanella ALDH16 is found to be a bona fide enzyme, exhibiting NAD+-binding, ALDH activity, and esterase activity. In contrast, human ALDH16A1 apparently lacks measurable aldehyde oxidation activity, suggesting that it is a pseudoenzyme, consistent with the absence of the catalytic Cys in its sequence. The fold of ALDH16 comprises three domains: NAD+-binding, catalytic, and C-terminal. The latter is unique to ALDH16 and features a Rossmann fold connected to a protruding β-flap. The tertiary structural interactions of the C-terminal domain mimic the quaternary structural interactions of the classic ALDH superfamily dimer, a phenomenon we call “trans-hierarchical structural similarity.” ALDH16 forms a unique dimer in solution, which mimics the classic ALDH superfamily dimer-of-dimer tetramer. Small-angle X-ray scattering shows that human ALDH16A1 has the same dimeric structure and fold as Loktanella ALDH16. We suggest that the Loktanella ALDH16 structure may be considered to be the archetype of the ALDH16 family.

Identification of a new nucleotide binding site by structural alignment and site directed mutagenesis.

Nucleotide binding proteins are involved in many important cellular processes and form one of the largest protein families. Traditionally, the identification of nucleotide binding motif, such as the ATP binding P-loop, has relied on the comparison of protein sequences, consideration of the function of each of the proteins and the identification of signature motifs within the sequence. Sometimes, it is difficult to identify nucleotide binding proteins based on sequence alignment because of increased evolutionary distances. In such cases, structural alignments can provide a better guide for comparing specific features of sequences because the overall structures of these motifs are conserved despite low sequence identity. In the present study, on the basis of bioinformatics and structural comparison of three representative protein structures of Ham1 superfamily, YjjX, YggV, and YhdE, previously identified as nucleotide binding proteins, we have identified a novel nucleotide binding motif (T/SXXXXK/R). The importance of this signature motif in binding of nucleotides was validated using site directed mutagenesis. Mutations of conserved residues of the loop either decreased or completely abolished the nucleotide binding activity of the protein. We used the conserved motif identified in the study to search for other proteins having a similar motif. Two proteins, GTP cyclohydrolase II and dephospho-CoA pyrophosphorylase showed presence of the loop, suggesting that this nucleotide binding motif is not unique in the Ham1 superfamily, but represents a novel NTP recognition motif.

A Type III-B Cmr effector complex catalyzes the synthesis of cyclic oligoadenylate second messengers by cooperative substrate binding.

Recently, Type III-A CRISPR-Cas systems were found to catalyze the synthesis of cyclic oligoadenylates (cOAs), a second messenger that specifically activates Csm6, a Cas accessory RNase and confers antiviral defense in bacteria. To test if III-B CRISPR-Cas systems could mediate a similar CRISPR signaling pathway, the Sulfolobus islandicus Cmr-α ribonucleoprotein complex (Cmr-α–RNP) was purified from the native host and tested for cOA synthesis. We found that the system showed a robust production of cyclic tetra-adenylate (c-A4), and that c-A4 functions as a second messenger to activate the III-B-associated RNase Csx1 by binding to its CRISPR-associated Rossmann Fold domain. Investigation of the kinetics of cOA synthesis revealed that Cmr-α–RNP displayed positively cooperative binding to the adenosine triphosphate (ATP) substrate. Furthermore, mutagenesis of conserved domains in Cmr2α confirmed that, while Palm 2 hosts the active site of cOA synthesis, Palm 1 domain serves as the primary site in the enzyme–substrate interaction. Together, our data suggest that the two Palm domains cooperatively interact with ATP molecules to achieve a robust cOA synthesis by the III-B CRISPR-Cas system.

The structure of monoamine oxidases: past, present, and future.

The first crystal structure of mammalian monoamine oxidases (MAOs) was solved in 2002; almost 65years after, these FAD-dependent enzymes were discovered and classified as responsible for the oxidation of aromatic neurotransmitters. Both MAO A and MAO B feature a two-domain topology characterized by the Rossmann fold, interacting with dinucleotide cofactors, which is intimately associated to a substrate-binding domain. This globular body is endowed with a C-terminal α-helix that anchors the protein to the outer mitochondrial phospholipid bilayer. As monotopic membrane proteins, the structural elucidation of MAOs was a challenging task that required the screening of different detergent conditions for their purification and crystallization. MAO A and MAO B structures differ both in their oligomerization architecture and in details of their active sites. Purified human MAO B and rat MAO A are dimeric, whereas human MAO A was found to be monomeric, which is believed to result from the detergent treatments used to extract the protein from the membrane. The active site of MAOs consists of a hydrophobic cavity located in front of the flavin cofactor and extending to the protein surface. Some structural features are highly conserved in the two isozymes, such as a Tyr-Tyr aromatic sandwich in front of the flavin ring and a Lys residue hydrogen-bonded to the cofactor N5 atom, whereas a pair of gating residues (Phe208/Ile335 in MAO A; Ile199/Tyr326 in MAO B) specifically determines the different substrate and inhibitor properties of the two enzymes.