Active Site Loop Research Articles

Structures of the alkanesulfonate monooxygenase MsuD provide insight into C–S bond cleavage, substrate scope, and an unexpected role for the tetramer

Bacterial two-component flavin-dependent monooxygenases cleave the stable C–S bond of environmental and anthropogenic organosulfur compounds. The monooxygenase MsuD converts methanesulfonate (MS−) to sulfite, completing the sulfur assimilation process during sulfate starvation, but the mechanism of this conversion remains unclear. To explore the mechanism of C–S bond cleavage, we report a series of crystal structures of MsuD from Pseudomonas fluorescens in different liganded states. This report provides the first crystal structures of an alkanesulfonate monooxygenase with a bound flavin and alkanesulfonate, elucidating the roles of the active site lid, the protein C terminus, and an active site loop in flavin and/or alkanesulfonate binding. These structures position MS− closest to the flavin N5 position, consistent with an N5-(hydro)peroxyflavin mechanism rather than a classical C4a-(hydro)peroxyflavin mechanism. A fully enclosed active site is observed in the ternary complex, mediated by interchain interaction of the C terminus at the tetramer interface. These structures identify an unexpected function of the protein C terminus in this protein family in stabilizing tetramer formation and the alkanesulfonate-binding site. Spurred by interest from the crystal structures, we conducted biochemical assays and molecular docking that redefine MsuD as a small- to medium-chain alkanesulfonate monooxygenase. Functional mutations verify the sulfonate-binding site and reveal the critical importance of the protein C terminus for monooxygenase function. These findings reveal a deeper understanding of MsuD’s functionality at the molecular level and consequently how it operates within its role as part of the sulfur assimilation pathway.

A higher flexibility at the SARS-CoV-2 main protease active site compared to SARS-CoV and its potentialities for new inhibitor virtual screening targeting multi-conformers

The main-protease (Mpro) catalyzes a crucial step for the SARS-CoV-2 life cycle. The recent SARS-CoV-2 presents the main protease (MCoV2 pro) with 12 mutations compared to SARS-CoV (MCoV1 pro). Recent studies point out that these subtle differences lead to mobility variances at the active site loops with functional implications. We use metadynamics simulations and a sort of computational analysis to probe the dynamic, pharmacophoric and catalytic environment differences between the monomers of both enzymes. So, we verify how much intrinsic distinctions are preserved in the functional dimer of MCoV2 pro, as well as its implications for ligand accessibility and optimized drug screening. We find a significantly higher accessibility to open binding conformers in the MCoV2 pro monomer compared to MCoV1 pro. A higher hydration propensity for the MCoV2 pro S2 loop with the A46S substitution seems to exercise a key role. Quantum calculations suggest that the wider conformations for MCoV2 pro are less catalytically active in the monomer. However, the statistics for contacts involving the N-finger suggest higher maintenance of this activity at the dimer. Docking analyses suggest that the ability to vary the active site width can be important to improve the access of the ligand to the active site in different ways. So, we carry out a multiconformational virtual screening with different ligand bases. The results point to the importance of taking into account the protein conformational multiplicity for new promissors anti MCoV2 pro ligands. We hope these results will be useful in prospecting, repurposing and/or designing new anti SARS-CoV-2 drugs. Communicated by Ramaswamy H. Sarma

Interrogation of Bacillus anthracis SrtA active site loop forming open/close lid conformations through extensive MD simulations for understanding binding selectivity of SrtA inhibitors.

Bacillus anthracis is a gram positive, deadly spore forming bacteria causing anthrax and these bacteria having the complex mechanism in the cell wall envelope, which can adopt the changes in environmental conditions. In this, the membrane bound cell wall proteins are said to progressive drug target for the inhibition of Bacillus anthracis. Among the cell wall proteins, the SrtA is one of the important mechanistic protein, which mediate the ligation with LPXTG motif by forming the amide bonds. The SrtA plays the vital role in cell signalling, cell wall formation, and biofilm formations. Inhibition of SrtA leads to rupture of the cell wall and biofilm formation, and that leads to inhibition of Bacillus anthracis and thus, SrtA is core important enzyme to study the inhibition mechanism. In this study, we have examined 28 compounds, which have the inhibitory activity against the Bacillus anthracis SrtA for developing the 3D-QSAR and also, compounds binding selectivity with both open and closed SrtA conformations, obtained from 100 ns of MD simulations. The binding site loop deviate in forming the open and closed gate mechanism is investigated to understand the inhibitory profile of reported compounds, and results show the closed state active site conformations are required for ligand binding specificity. Overall, the present study may offer an opportunity for better understanding of the mechanism of action and can be aided to further designing of a novel and highly potent SrtA inhibitors.

Initiation of Fatty Acid Synthesis by a Malonyl‐ACP Decarboxylase

Fatty acid synthesis is important in both prokaryotes and eukaryotes for formation of functional membranes and dysregulation of the pathway has implications for cell growth and survival. In bacteria, fatty acid production is tightly regulated by the Type II fatty acid synthesis (FASII) pathway. In Escherichia coli FabH (3-ketoacyl acyl carrier protein (ACP) synthase III) initiates fatty acid synthesis by condensing acetyl coenzyme A (acetyl-CoA) with malonyl-ACP. This work describes a new pathway enzyme, malonyl-ACP decarboxylase (MadA), that permits ΔfabH knockout strains to survive by providing an alternate pathway for the initiation of fatty acid synthesis. MadA has an N-terminal GCN5-related N-acetyltransferase (GNAT) domain and a C-terminal hotdog domain. The full-length protein, the N-terminal (MadAN) and C-terminal (MadAC) domains were purified. MadA and MadAC are dimers and MadAN is a monomer. MadAC is a malonyl-ACP decarboxylase and MadAN is not. Mad proteins are widely distributed in Proteobacteria and most contain only the C-terminal hot dog dimerization and catalytic domain (MadB). The MadB homolog in Shewanella oneidensis was purified. Site directed mutagenesis guided by the MadB crystal structure was used to identify the active site loop. Asn45 has a critical role in the malonyl-ACP decarboxylase activity. MadA initiates FabH-independent fatty acid synthesis by decarboxylating malonyl-ACP to acetyl-ACP. FabB/F then condenses malonyl-ACP and acetyl-ACP to acetoacetyl-ACP, which enters the elongation cycle. MadAC and MadB both restore the cell size defect in the ΔfabH strain.

Salmonella enterica BcfH Is a Trimeric Thioredoxin-Like Bifunctional Enzyme with Both Thiol Oxidase and Disulfide Isomerase Activities.

Aims: Thioredoxin (TRX)-fold proteins are ubiquitous in nature. This redox scaffold has evolved to enable a variety of functions, including redox regulation, protein folding, and oxidative stress defense. In bacteria, the TRX-like disulfide bond (Dsb) family mediates the oxidative folding of multiple proteins required for fitness and pathogenic potential. Conventionally, Dsb proteins have specific redox functions with monomeric and dimeric Dsbs exclusively catalyzing thiol oxidation and disulfide isomerization, respectively. This contrasts with the eukaryotic disulfide forming machinery where the modular TRX protein disulfide isomerase (PDI) mediates thiol oxidation and disulfide reshuffling. In this study, we identified and structurally and biochemically characterized a novel Dsb-like protein from Salmonella enterica termed bovine colonization factor protein H (BcfH) and defined its role in virulence. Results: In the conserved bovine colonization factor (bcf) fimbrial operon, the Dsb-like enzyme BcfH forms a trimeric structure, exceptionally uncommon among the large and evolutionary conserved TRX superfamily. This protein also displays very unusual catalytic redox centers, including an unwound α-helix holding the redox active site and a trans-proline instead of the conserved cis-proline active site loop. Remarkably, BcfH displays both thiol oxidase and disulfide isomerase activities contributing to Salmonella fimbrial biogenesis. Innovation and Conclusion: Typically, oligomerization of bacterial Dsb proteins modulates their redox function, with monomeric and dimeric Dsbs mediating thiol oxidation and disulfide isomerization, respectively. This study demonstrates a further structural and functional malleability in the TRX-fold protein family. BcfH trimeric architecture and unconventional catalytic sites permit multiple redox functions emulating in bacteria the eukaryotic PDI dual oxidoreductase activity. Antioxid. Redox Signal. 35, 21-39.

Active-site loop variations adjust activity and selectivity of the cumene dioxygenase

Active-site loops play essential roles in various catalytically important enzyme properties like activity, selectivity, and substrate scope. However, their high flexibility and diversity makes them challenging to incorporate into rational enzyme engineering strategies. Here, we report the engineering of hot-spots in loops of the cumene dioxygenase from Pseudomonas fluorescens IP01 with high impact on activity, regio- and enantioselectivity. Libraries based on alanine scan, sequence alignments, and deletions along with a novel insertion approach result in up to 16-fold increases in activity and the formation of novel products and enantiomers. CAVER analysis suggests possible increases in the active pocket volume and formation of new active-site tunnels, suggesting additional degrees of freedom of the substrate in the pocket. The combination of identified hot-spots with the Linker In Loop Insertion approach proves to be a valuable addition to future loop engineering approaches for enhanced biocatalysts.

Structure of a reaction intermediate mimic in t6A biosynthesis bound in the active site of the TsaBD heterodimer from Escherichia coli.

The tRNA modification N6-threonylcarbamoyladenosine (t6A) is universally conserved in all organisms. In bacteria, the biosynthesis of t6A requires four proteins (TsaBCDE) that catalyze the formation of t6A via the unstable intermediate l-threonylcarbamoyl-adenylate (TC-AMP). While the formation and stability of this intermediate has been studied in detail, the mechanism of its transfer to A37 in tRNA is poorly understood. To investigate this step, the structure of the TsaBD heterodimer from Escherichia coli has been solved bound to a stable phosphonate isosteric mimic of TC-AMP. The phosphonate inhibits t6A synthesis in vitro with an IC50 value of 1.3 μM in the presence of millimolar ATP and L-threonine. The inhibitor binds to TsaBD by coordination to the active site Zn atom via an oxygen atom from both the phosphonate and the carboxylate moieties. The bound conformation of the inhibitor suggests that the catalysis exploits a putative oxyanion hole created by a conserved active site loop of TsaD and that the metal essentially serves as a binding scaffold for the intermediate. The phosphonate bound crystal structure should be useful for the rational design of potent, drug-like small molecule inhibitors as mechanistic probes or potentially novel antibiotics.

Structural characterization of the family GH115 α-glucuronidase from Amphibacillus xylanus yields insight into its coordinated action with α-arabinofuranosidases

The coordinated action of carbohydrate-active enzymes has mainly been evaluated for the purpose of complete saccharification of plant biomass (lignocellulose) to sugars. By contrast, the coordinated action of accessory hemicellulases on xylan debranching and recovery is less well characterized. Here, the activity of two family GH115 α-glucuronidases (SdeAgu115A from Saccharophagus degradans, and AxyAgu115A from Amphibacillus xylanus) on spruce arabinoglucuronoxylan (AGX) was evaluated in combination with an α-arabinofuranosidase from families GH51 (AniAbf51A, aka E-AFASE from Aspergillus niger) and GH62 (SthAbf62A from Streptomyces thermoviolaceus). The α-arabinofuranosidases boosted (methyl)-glucuronic acid release by SdeAgu115A by approximately 50 % and 30 %, respectively. The impact of the α-arabinofuranosidases on AxyAgu115A activity was comparatively low, motivating its structural characterization. The crystal structure of AxyAgu115A revealed increased length and flexibility of the active site loop compared to SdeAgu115A. This structural difference could explain the ability of AxyAgu115A to accommodate more highly substituted arabinoglucuronoxylan, and inform enzyme selections for improved AGX recovery and use.

Water-mediated structural rearrangement establishes active conformation of caspases for apoptosis and inflammation

Caspases are cysteine-dependent aspartate-specific proteases that play a crucial role in apoptosis (or programmed cell death) and inflammation. Based on their function, caspases are majorly categorized into apoptotic (initiator/apical and effector/executioner) and inflammatory caspases. Caspases undergo transition from an inactive zymogen to an active caspase to accomplish their function. This transition demands structural rearrangements which are most prominent at the active site loops and are imperative for the catalytic activity of caspases. In effector caspase-3, the structural rearrangement in the active site loop is shown to be facilitated by a set of invariant water (IW) molecules. However, the atomic details involving their role in stabilizing the active conformation have not been reported yet. Moreover, it is not known whether water molecules are essential for the active conformation in all caspases. Thus, in this study, we located IW molecules in initiator, effector, and inflammatory caspases to understand their precise role in rendering the structural arrangement of active caspases. Furthermore, IW molecules involved in anchoring the fragments of the protomer and rendering regulated flaccidity to caspases were identified. Location and identification of IW molecules interacting with amino acid residues involved in establishing the active conformation in the caspases might facilitate the design of potent inhibitors during up-regulated caspase activity in neurodegenerative and immune disorders. Communicated by Ramaswamy H. Sarma

Identification of Thermal Conduits That Link the Protein-Water Interface to the Active Site Loop and Catalytic Base in Enolase.

We report here on the salient role of protein mobility in accessing conformational landscapes that enable efficient enzyme catalysis. We are focused on yeast enolase, a highly conserved lyase with a TIM barrel domain and catalytic loop, as part of a larger study of the relationship of site selective protein motions to chemical reactivity within superfamilies. Enthalpically hindered variants were developed by replacement of a conserved hydrophobic side chain (Leu 343) with smaller side chains. Leu343 is proximal to the active site base in enolase, and comparative pH rate profiles for the valine and alanine variants indicate a role for side chain hydrophobicity in tuning the pKa of the catalytic base. However, the magnitude of a substrate deuterium isotope effect is almost identical for wild-type (WT) and Leu343Ala, supporting an unchanged rate-determining proton abstraction step. The introduced hydrophobic side chains at position 343 lead to a discontinuous break in both activity and activation energy as a function of side chain volume. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments were performed as a function of time and temperature for WT and Leu343Ala, and provide a spatially resolved map of changes in protein flexibility following mutation. Impacts on protein flexibility are localized to specific networks that arise at the protein-solvent interface and terminate in a loop that has been shown by X-ray crystallography to close over the active site. These interrelated effects are discussed in the context of long-range, solvent-accessible and thermally activated networks that play key roles in tuning the precise distances and interactions among reactants.

Local interactions with the Glu166 base and the conformation of an active site loop play key roles in carbapenem hydrolysis by the KPC-2 β-lactamase

The Klebsiella pneumoniae carbapenemase-2 (KPC-2) is a common source of antibiotic resistance in Gram-negative bacterial infections. KPC-2 is a class A β-lactamase that exhibits a broad substrate profile and hydrolyzes most β-lactam antibiotics including carbapenems owing to rapid deacylation of the covalent acyl-enzyme intermediate. However, the features that allow KPC-2 to deacylate substrates more rapidly than non-carbapenemase enzymes are not clear. The active-site residues in KPC-2 are largely conserved in sequence and structure compared with non-carbapenemases, suggesting that subtle alterations may collectively facilitate hydrolysis of carbapenems. We utilized a nonbiased genetic approach to identify mutants deficient in carbapenem hydrolysis but competent for ampicillin hydrolysis. Subsequent pre–steady-state enzyme kinetics analyses showed that the substitutions slow the rate of deacylation of carbapenems. Structure determination via X-ray diffraction indicated that a F72Y mutant forms a hydrogen bond between the tyrosine hydroxyl group and Glu166, which may lower basicity and impair the activation of the catalytic water for deacylation, whereas several mutants impact the structure of the Q214-R220 active site loop. A T215P substitution lowers the deacylation rate and drastically alters the conformation of the loop, thereby disrupting interactions between the enzyme and the carbapenem acyl-enzyme intermediate. Thus, the environment of the Glu166 general base and the precise placement and conformational stability of the Q214-R220 loop are critical for efficient deacylation of carbapenems by the KPC-2 enzyme. Therefore, the design of carbapenem antibiotics that interact with Glu166 or alter the Q214-R220 loop conformation may disrupt enzyme function and overcome resistance.

Allosteric and dynamic control of RNA-dependent RNA polymerase function and fidelity.

All RNA viruses encode an RNA-dependent RNA polymerase (RdRp) responsible for genome replication. It is now recognized that enzymes in general, and RdRps specifically, are dynamic macromolecular machines such that their moving parts, including active site loops, play direct functional roles. While X-ray crystallography has provided deep insight into structural elements important for RdRp function, this methodology generally provides only static snapshots, and so is limited in its ability to report on dynamic fluctuations away from the lowest energy conformation. Nuclear magnetic resonance (NMR), molecular dynamics (MD) simulations and other biophysical techniques have brought new insight into RdRp function by their ability to characterize the trajectories, kinetics and thermodynamics of conformational motions. In particular, these methodologies have identified coordinated motions among conserved structural motifs necessary for nucleotide selection and incorporation. Disruption of these motions through amino acid substitutions or inhibitor binding impairs RdRp function. Understanding and re-engineering these motions thus provides exciting new avenues for anti-viral strategies. This chapter outlines the basics of these methodologies, summarizes the dynamic motions observed in different RdRps important for nucleotide selection and incorporation, and illustrates how this information can be leveraged towards rational vaccine strain development and anti-viral drug design.

Conformational dynamics is critical for the allosteric inhibition of cGAS upon acetyl-mimic mutations.

Detection of cytosolic dsDNA by cyclic GMP-AMP synthase (cGAS) is critical for the immune system to sense and fight against infection, but chronic activation of cGAS by self-DNA leads to autoimmune diseases without effective treatment yet. It was found that acetylation on either Lys384, Lys394, or Lys414 could inhibit the catalytic production of cGAMP by cGAS, and further suppressed self-DNA-induced autoimmunity. However, the implied mechanism remains unclear. Here, extensive molecular dynamics simulations combined with multiple analytical approaches were employed to uncover the allosteric inhibition mechanisms by using the K-to-Q mutations to mimic acetylation. Results suggested that the exterior loops contributed most to the conformational dynamics of cGAS, and two concerted intrinsic motions were observed: the inward/outward or twisting movement for the outer appendage of lobe 1 and the open/closed swing of the active-site loops. Mutations slightly affected the binding of dsDNA and cGAMP. The shift of the conformational sampling of the active-site loops or residues around cGAMP upon mutation might potentially explain the inhibition of cGAS activity. Moreover, the intra- and inter-molecular coupling was weakened upon mutations more or less but via distinct pathways. Hence, conformational dynamics play a vital role in the allosteric inhibition of cGAS upon the studied acetyl-mimic mutations. As the studied acetyl-mimic mutations are located at either the inter-lobe or inter-molecular interfaces, hence except for acetylation, our findings might help the development of new therapeutics against autoimmune diseases due to abnormal cGAS activation by designing inter-lobe or intermolecular allosteric inhibitors.

Conformational Plasticity in Human Heme-Based Dioxygenases.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and human tryptophan dioxygenase (hTDO) are two important heme proteins that degrade the essential amino acid, l-tryptophan (Trp), along the kynurenine pathway. The two enzymes share a similar active site structure and an analogous catalytic mechanism, but they exhibit a variety of distinct functional properties. Here we used carbon monoxide (CO) as a structural probe to interrogate how the functionalities of the two enzymes are encoded in their structures. With X-ray crystallography, we detected an unexpected photochemical intermediate trapped in a crystal of the hIDO1-CO-Trp complex, where CO is photolyzed from the heme iron by X-rays at cryogenic temperatures (100 K). The CO photolysis triggers a large-scale migration of the substrate Trp, as well as the photolyzed CO, from the active site to a temporary binding site, Sa*. It is accompanied by a large conformational change to an active site loop, JK-LoopC, despite the severely restricted protein motion under the frozen conditions, which highlights the remarkable conformational plasticity of the hIDO1 protein. Comparative studies of a crystal of the hTDO-CO-Trp complex show that CO and Trp remain bound in the active site under comparable X-ray illumination, indicating a much more rigid protein architecture. The data offer important new insights into the structure and function relationships of the heme-based dioxygenases and provide new guidelines for structure-based design of inhibitors targeting them.

Structure and Characterization of Phosphoglucomutase 5 from Atlantic and Baltic Herring-An Inactive Enzyme with Intact Substrate Binding.

Phosphoglucomutase 5 (PGM5) in humans is known as a structural muscle protein without enzymatic activity, but detailed understanding of its function is lacking. PGM5 belongs to the alpha-D-phosphohexomutase family and is closely related to the enzymatically active metabolic enzyme PGM1. In the Atlantic herring, Clupea harengus, PGM5 is one of the genes strongly associated with ecological adaptation to the brackish Baltic Sea. We here present the first crystal structures of PGM5, from the Atlantic and Baltic herring, differing by a single substitution Ala330Val. The structure of PGM5 is overall highly similar to structures of PGM1. The structure of the Baltic herring PGM5 in complex with the substrate glucose-1-phosphate shows conserved substrate binding and active site compared to human PGM1, but both PGM5 variants lack phosphoglucomutase activity under the tested conditions. Structure comparison and sequence analysis of PGM5 and PGM1 from fish and mammals suggest that the lacking enzymatic activity of PGM5 is related to differences in active-site loops that are important for flipping of the reaction intermediate. The Ala330Val substitution does not alter structure or biophysical properties of PGM5 but, due to its surface-exposed location, could affect interactions with protein-binding partners.

HEPN-MNT Toxin-Antitoxin System: The HEPN Ribonuclease Is Neutralized by OligoAMPylation

Prokaryotic toxin-antitoxin (TA) systems are composed of a toxin capable of interfering with key cellular processes and its neutralizing antidote, the antitoxin. Here, we focus on the HEPN-MNT TA system encoded in the vicinity of a subtype I-D CRISPR-Cas system in the cyanobacterium Aphanizomenon flos-aquae. We show that HEPN acts as a toxic RNase, which cleaves off 4 nt from the 3' end in a subset of tRNAs, thereby interfering with translation. Surprisingly, we find that the MNT (minimal nucleotidyltransferase) antitoxin inhibits HEPN RNase through covalent di-AMPylation (diadenylylation) of a conserved tyrosine residue, Y109, in the active site loop. Furthermore, we present crystallographic snapshots of the di-AMPylation reaction at different stages that explain the mechanism of HEPN RNase inactivation. Finally, we propose that the HEPN-MNT system functions as a cellular ATP sensor that monitors ATP homeostasis and, at low ATP levels, releases active HEPN toxin.

Characterizing human α-1,6-fucosyltransferase (FUT8) substrate specificity and structural similarities with related fucosyltransferases

Mammalian Asn-linked glycans are extensively processed as they transit the secretory pathway to generate diverse glycans on cell surface and secreted glycoproteins. Additional modification of the glycan core by α-1,6-fucose addition to the innermost GlcNAc residue (core fucosylation) is catalyzed by an α-1,6-fucosyltransferase (FUT8). The importance of core fucosylation can be seen in the complex pathological phenotypes of FUT8 null mice, which display defects in cellular signaling, development, and subsequent neonatal lethality. Elevated core fucosylation has also been identified in several human cancers. However, the structural basis for FUT8 substrate specificity remains unknown.Here, using various crystal structures of FUT8 in complex with a donor substrate analog, and with four distinct glycan acceptors, we identify the molecular basis for FUT8 specificity and activity. The ordering of three active site loops corresponds to an increased occupancy for bound GDP, suggesting an induced-fit folding of the donor-binding subsite. Structures of the various acceptor complexes were compared with kinetic data on FUT8 active site mutants and with specificity data from a library of glycan acceptors to reveal how binding site complementarity and steric hindrance can tune substrate affinity. The FUT8 structure was also compared with other known fucosyltransferases to identify conserved and divergent structural features for donor and acceptor recognition and catalysis. These data provide insights into the evolution of modular templates for donor and acceptor recognition among GT-B fold glycosyltransferases in the synthesis of diverse glycan structures in biological systems.

Structure and dynamics of an \u03b1-fucosidase reveal \ta mechanism for highly efficient IgG transfucosylation

Fucosylation is important for the function of many proteins with biotechnical and medical applications. Alpha-fucosidases comprise a large enzyme family that recognizes fucosylated substrates with diverse α-linkages on these proteins. Lactobacillus casei produces an α-fucosidase, called AlfC, with specificity towards α(1,6)-fucose, the only linkage found in human N-glycan core fucosylation. AlfC and certain point mutants thereof have been used to add and remove fucose from monoclonal antibody N-glycans, with significant impacts on their effector functions. Despite the potential uses for AlfC, little is known about its mechanism. Here, we present crystal structures of AlfC, combined with mutational and kinetic analyses, hydrogen–deuterium exchange mass spectrometry, molecular dynamic simulations, and transfucosylation experiments to define the molecular mechanisms of the activities of AlfC and its transfucosidase mutants. Our results indicate that AlfC creates an aromatic subsite adjacent to the active site that specifically accommodates GlcNAc in α(1,6)-linkages, suggest that enzymatic activity is controlled by distinct open and closed conformations of an active-site loop, with certain mutations shifting the equilibrium towards open conformations to promote transfucosylation over hydrolysis, and provide a potentially generalizable framework for the rational creation of AlfC transfucosidase mutants.

Structure and substrate specificity determinants of the taurine biosynthetic enzyme cysteine sulphinic acid decarboxylase

Pyridoxal 5́-phosphate (PLP) is an important cofactor for amino acid decarboxylases with many biological functions, including the synthesis of signalling molecules, such as serotonin, dopamine, histamine, γ-aminobutyric acid, and taurine. Taurine is an abundant amino acid with multiple physiological functions, including osmoregulation, pH regulation, antioxidative protection, and neuromodulation. In mammalian tissues, taurine is mainly produced by decarboxylation of cysteine sulphinic acid to hypotaurine, catalysed by the PLP-dependent cysteine sulphinic acid decarboxylase (CSAD), followed by oxidation of the product to taurine. We determined the crystal structure of mouse CSAD and compared it to other PLP-dependent decarboxylases in order to identify determinants of substrate specificity and catalytic activity. Recognition of the substrate involves distinct side chains forming the substrate-binding cavity. In addition, the backbone conformation of a buried active-site loop appears to be a critical determinant for substrate side chain binding in PLP-dependent decarboxylases. Phe94 was predicted to affect substrate specificity, and its mutation to serine altered both the catalytic properties of CSAD and its stability. Using small-angle X-ray scattering, we further showed that CSAD presents open/close motions in solution. The structure of apo-CSAD indicates that the active site gets more ordered upon internal aldimine formation. Taken together, the results highlight details of substrate recognition in PLP-dependent decarboxylases and provide starting points for structure-based inhibitor design with the aim of affecting the biosynthesis of taurine and other abundant amino acid metabolites.

Structural and functional analyses of the PPIase domain of Arabidopsisthaliana CYP71 reveal its catalytic activity toward histone H3.

Arabidopsisthaliana CYP71 (AtCYP71) is a chromatin-remodeling protein that promotes shoot apical meristem (SAM) differentiation. The N terminus of AtCYP71 contains a noncanonical WD domain, and the C terminus contains an enzymatic peptidyl-prolyl isomerase (PPIase) cyclophilin (CYP) domain. To date, there has been no characterization of CYP71, and its mode of action remains unknown. Here, we report the crystal structure of the CYP domain of AtCYP71 at 1.9Å resolution. The structure shows key differences when compared to the canonical CYP fold of human CypA. To the best our knowledge, this is the first A.thaliana CYP structure with a conserved active site loop. Using nuclear magnetic resonance spectroscopy, we demonstrate that the CYP domain is active toward histone H3. Our findings suggest that the PPIase activity of the CYP domain is important for the function of AtCYP71 in chromatin remodeling during organogenesis.