Rossmann-fold Proteins Research Articles

Putative NAD(P)-Binding Rossmann Fold Protein Is Involved in Chitosan-Induced Peroxidase Activity and Lipoxygenase Expression in Physcomitrium patens.

Oxidative burst, the rapid production of high levels of reactive oxygen species in response to external stimuli, is an early defense reaction against pathogens. The fungal elicitor chitosan causes an oxidative burst in the moss Physcomitrium patens (formerly Physcomitrella patens), mainly due to the peroxidase enzyme Prx34. To better understand the chitosan responses in P. patens, we conducted a screen of part of a P. patens mutant collection to isolate plants with less peroxidase activity than wild-type (WT) plants after chitosan treatment. We isolated a P.patens mutant that affected the gene encoding NAD(P)-binding Rossmann fold protein (hereafter, Rossmann fold protein). Three Rossmann fold protein-knockout (KO) plants (named Rossmann fold KO lines) were generated and used to assess extracellular peroxidase activity and expression of defense-responsive genes, including alternative oxidase, lipoxygenase (LOX), NADPH oxidase, and peroxidase (Prx34) in response to chitosan treatment. Extracellular (apoplastic) peroxidase activity was significantly lower in Rossmann fold KO lines than in WT plants after chitosan treatments. Expression of the LOX gene in Rossmann fold KO plants was significantly lower before and after chitosan treatment when compared with WT. Peroxidase activity assays together with gene expression analyses suggest that the Rossmann fold protein might be an important component of the signaling pathway leading to oxidative burst and basal expression of the LOX gene in P. patens. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

ROS, Moss, and a Rossmann Fold Protein: Identification of a Key Signaling Component for Plant Immunity.

ROS, Moss, and a Rossmann Fold Protein: Identification of a Key Signaling Component for Plant Immunity.

The RNA-bound proteome of MRSA reveals post-transcriptional roles for helix-turn-helix DNA-binding and Rossmann-fold proteins

RNA-binding proteins play key roles in controlling gene expression in many organisms, but relatively few have been identified and characterised in detail in Gram-positive bacteria. Here, we globally analyse RNA-binding proteins in methicillin-resistant Staphylococcus aureus (MRSA) using two complementary biochemical approaches. We identify hundreds of putative RNA-binding proteins, many containing unconventional RNA-binding domains such as Rossmann-fold domains. Remarkably, more than half of the proteins containing helix-turn-helix (HTH) domains, which are frequently found in prokaryotic transcription factors, bind RNA in vivo. In particular, the CcpA transcription factor, a master regulator of carbon metabolism, uses its HTH domain to bind hundreds of RNAs near intrinsic transcription terminators in vivo. We propose that CcpA, besides acting as a transcription factor, post-transcriptionally regulates the stability of many RNAs.

Rossmann-toolbox: a deep learning-based protocol for the prediction and design of cofactor specificity in Rossmann fold proteins.

The Rossmann fold enzymes are involved in essential biochemical pathways such as nucleotide and amino acid metabolism. Their functioning relies on interaction with cofactors, small nucleoside-based compounds specifically recognized by a conserved βαβ motif shared by all Rossmann fold proteins. While Rossmann methyltransferases recognize only a single cofactor type, the S-adenosylmethionine, the oxidoreductases, depending on the family, bind nicotinamide (nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate) or flavin-based (flavin adenine dinucleotide) cofactors. In this study, we showed that despite its short length, the βαβ motif unambiguously defines the specificity towards the cofactor. Following this observation, we trained two complementary deep learning models for the prediction of the cofactor specificity based on the sequence and structural features of the βαβ motif. A benchmark on two independent test sets, one containing βαβ motifs bearing no resemblance to those of the training set, and the other comprising 38 experimentally confirmed cases of rational design of the cofactor specificity, revealed the nearly perfect performance of the two methods. The Rossmann-toolbox protocols can be accessed via the webserver at https://lbs.cent.uw.edu.pl/rossmann-toolbox and are available as a Python package at https://github.com/labstructbioinf/rossmann-toolbox.

Physiological and proteomic analysis reveals the impact of boron deficiency and surplus on alfalfa (Medicago sativa L.) reproductive organs

Boron (B), an essential element for increasing seed yield and germinability in alfalfa (Medicago sativa L.), plays a vital role in its reproductive processes. However, effects of B stress on physiological and proteomic changes in reproductive organs related to alfalfa seed yield and germinability are poorly understood. In order to gain a better insight into B response or tolerance mechanisms, field trials were designed for B deficiency (0 mg B L−1), B sufficiency (800 mg B L−1), and B surplus (1600 mg B L−1) application during alfalfa flowering to analyze the proteomics and physiological responses of alfalfa ‘Aohan’ reproductive organs. Results showed that B deficiency weakened the stress-responsive ability in these organs, while B surplus reduced the sugar utilization of ‘Aohan’ flowers and caused lipid membrane peroxidation in ‘Aohan’ seeds. In addition, four upregulated stress responsive proteins (ADF-like protein, IMFP, NAD(P)-binding Rossmann-fold protein and NAD-dependent ALDHs) might play pivotal roles in the response of ‘Aohan’ reproductive organs to conditions of B deficiency and B surplus. All of the above results would be helpful to understand the tolerance mechanisms of alfalfa reproductive organs to both B deficiency and B surplus conditions, and also to give insight into the regulatory role of B in improving seed yield and germinability in alfalfa seed production. In summary, B likely plays a structural and regulatory role in relation to lipid metabolism, carbohydrate metabolism, amino acid metabolism, and signal transduction, thus regulates alfalfa reproductive processes eventually affecting the seed yield and germinability of alfalfa seeds.

Discovery of a Potent and Selective Coactivator Associated Arginine Methyltransferase 1 (CARM1) Inhibitor by Virtual Screening

Protein arginine methyltransferases (PRMTs) represent an emerging target class in oncology and other disease areas. So far, the most successful strategy to identify PRMT inhibitors has been to screen large to medium-size chemical libraries. Attempts to develop PRMT inhibitors using receptor-based computational methods have met limited success. Here, using virtual screening approaches, we identify 11 CARM1 (PRMT4) inhibitors with ligand efficiencies ranging from 0.28 to 0.84. CARM1 selective hits were further validated by orthogonal methods. Two structure-based rounds of optimization produced 27 (SGC2085), a CARM1 inhibitor with an IC50 of 50 nM and more than hundred-fold selectivity over other PRMTs. These results indicate that virtual screening strategies can be successfully applied to Rossmann-fold protein methyltransferases.

Characterization and Structure of the Aquifex aeolicus Protein DUF752: A BACTERIAL tRNA-METHYLTRANSFERASE (MnmC2) FUNCTIONING WITHOUT THE USUALLY FUSED OXIDASE DOMAIN (MnmC1)

Post-transcriptional modifications of the wobble uridine (U34) of tRNAs play a critical role in reading NNA/G codons belonging to split codon boxes. In a subset of Escherichia coli tRNA, this wobble uridine is modified to 5-methylaminomethyluridine (mnm(5)U34) through sequential enzymatic reactions. Uridine 34 is first converted to 5-carboxymethylaminomethyluridine (cmnm(5)U34) by the MnmE-MnmG enzyme complex. The cmnm(5)U34 is further modified to mnm(5)U by the bifunctional MnmC protein. In the first reaction, the FAD-dependent oxidase domain (MnmC1) converts cmnm(5)U into 5-aminomethyluridine (nm(5)U34), and this reaction is immediately followed by the methylation of the free amino group into mnm(5)U34 by the S-adenosylmethionine-dependent domain (MnmC2). Aquifex aeolicus lacks a bifunctional MnmC protein fusion and instead encodes the Rossmann-fold protein DUF752, which is homologous to the methyltransferase MnmC2 domain of Escherichia coli MnmC (26% identity). Here, we determined the crystal structure of the A. aeolicus DUF752 protein at 2.5 Å resolution, which revealed that it catalyzes the S-adenosylmethionine-dependent methylation of nm(5)U in vitro, to form mnm(5)U34 in tRNA. We also showed that naturally occurring tRNA from A. aeolicus contains the 5-mnm group attached to the C5 atom of U34. Taken together, these results support the recent proposal of an alternative MnmC1-independent shortcut pathway for producing mnm(5)U34 in tRNAs.

Automated hierarchical classification of protein domain subfamilies based on functionally-divergent residue signatures

BackgroundThe NCBI Conserved Domain Database (CDD) consists of a collection of multiple sequence alignments of protein domains that are at various stages of being manually curated into evolutionary hierarchies based on conserved and divergent sequence and structural features. These domain models are annotated to provide insights into the relationships between sequence, structure and function via web-based BLAST searches.ResultsHere we automate the generation of conserved domain (CD) hierarchies using a combination of heuristic and Markov chain Monte Carlo (MCMC) sampling procedures and starting from a (typically very large) multiple sequence alignment. This procedure relies on statistical criteria to define each hierarchy based on the conserved and divergent sequence patterns associated with protein functional-specialization. At the same time this facilitates the sequence and structural annotation of residues that are functionally important. These statistical criteria also provide a means to objectively assess the quality of CD hierarchies, a non-trivial task considering that the protein subgroups are often very distantly related—a situation in which standard phylogenetic methods can be unreliable. Our aim here is to automatically generate (typically sub-optimal) hierarchies that, based on statistical criteria and visual comparisons, are comparable to manually curated hierarchies; this serves as the first step toward the ultimate goal of obtaining optimal hierarchical classifications. A plot of runtimes for the most time-intensive (non-parallelizable) part of the algorithm indicates a nearly linear time complexity so that, even for the extremely large Rossmann fold protein class, results were obtained in about a day.ConclusionsThis approach automates the rapid creation of protein domain hierarchies and thus will eliminate one of the most time consuming aspects of conserved domain database curation. At the same time, it also facilitates protein domain annotation by identifying those pattern residues that most distinguish each protein domain subgroup from other related subgroups.

Protein Flexibility and Energy Flow During Enzyme Catalysis

Enzymes are dynamic molecules. Although in the past enzymes were viewed as static entities, recent evidence from experimental, theoretical and computational work indicates that protein dynamics play a significant role in enhancing catalytic activity. Investigations of the free energy profile for several proteins such as cyclophilin A and dihydrofolate reductase have revealed a network of protein motions that promote catalytic activity. Results indicate that these reaction-promoting motions are conserved as part of the enzyme fold across several species, even though they have low sequence similarity. Extending our study to a superfamily of enzymes, namely the dinucleotide binding Rossmann Fold proteins (DBRP), shows that in spite of having very low sequence homology and different structural features, the overall intrinsic dynamical flexibility of the superfamily is remarkably well preserved with respect to the catalytic step. The conformational coupling observed between exterior surface regions with the active site entails energetic coupling between them. To characterize this energetic coupling, we use an integrated information theoretic and biophysical approach to analyze residues that may constitute pathways through which energy may propagate from the flexible exterior surface regions to the active site of the protein. Our results reveal significant similarities in the energy flow pathways within the DBRP super-family. This study provides specific insights into how the DBRP super-family of proteins has evolved to catalyze hydride transfer reactions.

Identification in Agrobacterium tumefaciens of the d-galacturonic acid dehydrogenase gene

There are at least three different pathways for the catabolism of D-galacturonate in microorganisms. In the oxidative pathway, which was described in some prokaryotic species, D-galacturonate is first oxidised to meso-galactarate (mucate) by a nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase (EC 1.1.1.203). In the following steps of the pathway mucate is converted to 2-keto-glutarate. The enzyme activities of this catabolic pathway have been described while the corresponding gene sequences are still unidentified. The D-galacturonate dehydrogenase was purified from Agrobacterium tumefaciens, and the mass of its tryptic peptides was determined using MALDI-TOF mass spectrometry. This enabled the identification of the corresponding gene udh. It codes for a protein with 267 amino acids having homology to the protein family of NAD(P)-binding Rossmann-fold proteins. The open reading frame was functionally expressed in Saccharomyces cerevisiae. The N-terminally tagged protein was not compromised in its activity and was used after purification for a kinetic characterization. The enzyme was specific for NAD and accepted D-galacturonic acid and D-glucuronic acid as substrates with similar affinities. NMR analysis showed that in water solution the substrate D-galacturonic acid is predominantly in pyranosic form which is converted by the enzyme to 1,4 lactone of galactaric acid. This lactone seems stable under intracellular conditions and does not spontaneously open to the linear meso-galactaric acid.

Conserved Protein Flexibility And Pathways Of Energy Flow In Enzyme Catalysis

Conformational fluctuations in enzymes have significant affect on catalysis. Several enzymes show the presence of a network of coupled motions associated with the catalytic step. Here, we describe our recent studies to identify and characterize coupled motions in members of a diverse family of enzymes namely the dinucleotide binding Rossmann fold proteins (DBRP), sharing a common sub-step of hydride transfer from the dinucleotide cofactor to the substrate.Results show that in spite of low sequence/structural homology, the overall intrinsic dynamical flexibility during the course of the enzyme reaction is conserved. These dynamical fluctuations span from the exterior surface regions to the active site of the protein and form pathways. These pathways are connected via hydrogen bonds/hydrophobic interactions, which are conserved across prokaryotes and eukaryotes alike.In order to characterize the energy flow within these pathways, we use an integrated information theoretic and biophysical approach to study how energy may propagate within the DBRP super-family. The studies reveal for the first time how energy is propagated from the exterior flexible surface regions of the protein to the active site of the protein.

Prediction of coenzyme specificity in dehydrogenases/ reductases

Dehydrogenases and reductases are enzymes of fundamental metabolic importance that often adopt a specific structure known as the Rossmann fold. This fold, consisting of a six-stranded beta-sheet surrounded by alpha-helices, is responsible for coenzyme binding. We have developed a method to identify Rossmann folds and predict their coenzyme specificity (NAD, NADP or FAD) using only the amino acid sequence as input. The method is based upon hidden Markov models and sequence pattern analysis. The prediction sensitivity is 79% and the selectivity close to 100%. The method was applied on a set of 68 genomes, representing the three kingdoms archaea, bacteria and eukaryota. In prokaryotes, 3% of the genes were found to code for Rossmann-fold proteins, while the corresponding ratio in eukaryotes is only around 1%. In all genomes, NAD is the most preferred cofactor (41-49%), followed by NADP with 30-38%, while FAD is the least preferred cofactor (21%). However, the NAD preponderance over NADP is most pronounced in archaea, and least in eukaryotes. In all three kingdoms, only 3-8% of the Rossmann proteins are predicted to have more than one membrane-spanning segment, which is much lower than the frequency of membrane proteins in general. Analysis of the major protein types in eukaryotes reveals that the most common type (26%) of the Rossmann proteins are short-chain dehydrogenases/reductases. In addition, the identified Rossmann proteins were analyzed with respect to further protein types, enzyme classes and redundancy. The described method is available at http://www.ifm.liu.se/bioinfo, where the preferred coenzyme and its binding region are predicted given an amino acid sequence as input.

A new family of NAD(P)H-dependent oxidoreductases distinct from conventional Rossmann-fold proteins

A new family of NAD(P)H-dependent oxidoreductases is now recognized as a protein family distinct from conventional Rossmann-fold proteins. Numerous putative proteins belonging to the family have been annotated as malate dehydrogenase (MDH) or lactate dehydrogenase (LDH) according to the previous classification as type-2 malate/L-lactate dehydrogenases. However, recent biochemical and genetic studies have revealed that the protein family consists of a wide variety of enzymes with unique catalytic activities other than MDH or LDH activity. Based on their sequence homologies and plausible functions, the family proteins can be grouped into eight clades. This classification would be useful for reliable functional annotation of the new family of NAD(P)H-dependent oxidoreductases.

Structure of alanine dehydrogenase from Archaeoglobus: active site analysis and relation to bacterial cyclodeaminases and mammalian mu crystallin.

The hyperthermophilic archaeon Archaeoglobus fulgidus contains an L-Ala dehydrogenase (AlaDH, EC 1.4.1.1) that is not homologous to known bacterial dehydrogenases and appears to represent a previously unrecognized archaeal group of NAD-dependent dehydrogenases. The gene (Genbank; TIGR AF1665) was annotated initially as an ornithine cyclodeaminase (OCD) on the basis of strong homology with the mu crystallin/OCD protein family. We report the structure of the NAD-bound AF1665 AlaDH (AF-AlaDH) at 2.3 A in a C2 crystal form with the 70 kDa dimer in the asymmetric unit, as the first structural representative of this family. Consistent with its lack of homology to bacterial AlaDH proteins, which are mostly hexameric, the archaeal dimer has a novel structure. Although both types of AlaDH enzyme include a Rossmann-type NAD-binding domain, the arrangement of strands in the C-terminal half of this domain is novel, and the other (catalytic) domain in the archaeal protein has a new fold. The active site presents a cluster of conserved Arg and Lys side-chains over the pro-R face of the cofactor. In addition, the best ordered of the 338 water molecules in the structure is positioned well for mechanistic interaction. The overall structure and active site are compared with other dehydrogenases, including the AlaDH from Phormidium lapideum. Implications for the catalytic mechanism and for the structures of homologs are considered. The archaeal AlaDH represents an ancient and previously undescribed subclass of Rossmann-fold proteins that includes bacterial ornithine and lysine cyclodeaminases, marsupial lens proteins and, in man, a thyroid hormone-binding protein that exhibits 30% sequence identity with AF1665.

Structure of the RCK Domain from the E. coli K + Channel and Demonstration of Its Presence in the Human BK Channel

The intracellular C-terminal domain structure of a six-transmembrane K + channel from Escherichia coli has been solved by X-ray crystallography at 2.4 Å resolution. The structure is representative of a broad class of domains/proteins that regulate the conductance of K + (here referred to as RCK domains) in prokaryotic K + transporters and K + channels. The RCK domain has a Rossmann-fold topology with unique positions, not commonly conserved among Rossmann-fold proteins, composing a well-conserved salt bridge and a hydrophobic dimer interface. Structure-based amino acid sequence alignments and mutational analysis are used to demonstrate that an RCK domain is also present and is an important component of the gating machinery in eukaryotic large-conductance Ca 2+-activated K + channels.