Catalytic Machinery Research Articles

A high-speed network of nanoswitches for on/off control of catalysis.

NetState I of the communication-catalysis protocol is defined by a 1 : 1 mixture of the nanoswitches [Cu(1)]+ and 2. Upon one-electron oxidation at the ferrocenyl unit of the switch [Cu(1)]+, copper(i) ions are released that after translocation toggle nanoswitch 2 → [Cu(2)]+ (NetState II) within 4 min. NetState I was fully reset within 1 min by reduction of 1+ → 1. Running this redox-triggered switching protocol in the presence of 4-nitrobenzaldehyde, diethyl malonate and piperidine (catalyst) allows toggling of a catalyzed Knoevenagel addition from ON to OFF and back to ON.

Networking switches for smart functions using copper signaling and dynamic heteroleptic complexation.

This personal frontier account describes our recent progress in networking nanoswitches to generate emergent functions, such as catalytic machinery, and identifies the key impediments in mastering the paradigm shift from pure compounds to smart mixtures. A crucial challenge is the setup of reliable signaling protocols that are based on highly selective metal ion translocation and metal-ligand receptor events.

Coordinated Network Changes Across the Catalytic Cycle of Alpha Tryptophan Synthase

Networks of noncovalent interactions play important roles in the structural dynamics of globular proteins. Allosteric signals can propagate from the surface of an enzyme into its active site through these amino acid interaction networks. We used nuclear magnetic resonance chemical shift covariance analyses on a catalytically inactive variant of the alpha subunit of tryptophan synthase to map its amino acid interaction networks across its entire catalytic cycle. Each enzyme intermediate has unique sets of residues that display high chemical shift correlations across a set of alanine-to-glycine loop substitutions. Moreover, these chemical shift correlations systematically increase or decrease as a function of the catalytic cycle. These studies indicate that the enzyme intermediates have different amino acid interaction networks, and these network transitions are likely important for catalytic cycle progression. The different network interactions likely help to position catalytic machinery and coordinate function with the beta subunit.

Inhibitors against Fungal Cell Wall Remodeling Enzymes.

Fungal β-1,3-glucan glucanosyltransferases are glucan-remodeling enzymes that play important roles in cell wall integrity, and are essential for the viability of pathogenic fungi and yeasts. As such, they are considered possible drug targets, although inhibitors of this class of enzymes have not yet been reported. Herein we report a multidisciplinary approach based on a structure-guided design using a highly conserved transglycosylase from Sacharomyces cerevisiae, that leads to carbohydrate derivatives with high affinity for Aspergillus fumigatus Gel4. We demonstrate by X-ray crystallography that the compounds bind in the active site of Gas2/Gel4 and interact with the catalytic machinery. The topological analysis of noncovalent interactions demonstrates that the combination of a triazole with positively charged aromatic moieties are important for optimal interactions with Gas2/Gel4 through unusual pyridinium cation-π and face-to-face π-π interactions. The lead compound is capable of inhibiting AfGel4 with an IC50 value of 42 μm.

Four-Component Catalytic Machinery: Reversible Three-State Control of Organocatalysis by Walking Back and Forth on a Track.

A three-component supramolecular walker system is presented where a two-footed ligand (biped) walks back and forth on a tetrahedral 3D track upon the addition and removal of copper(I) ions, respectively. The addition of N-methylpyrrolidine as a catalyst to the walker system generates a four-component catalytic machinery, which acts as a three-state switchable catalytic ensemble in the presence of substrates for a conjugate addition. The copper(I)-ion-initiated walking process of the biped ligand on the track regulates the catalytic activity in three steps: ON versus intON (intermediate ON) versus OFF. To establish the operation of the four-component catalytic machinery in a mixture of all constituents, forward and backward cycles were performed in situ illustrating that both the walking process and catalytic action are fully reversible and reproducible.

Catalytic Three-Component Machinery: Control of Catalytic Activity by Machine Speed.

Three supramolecular slider-on-deck systems DS1-DS3 were obtained as two-component aggregates from the sliders S1-S3 and deck D with its three zinc porphyrin (ZnPor) binding sites. The binding of the two-footed slider to the deck varies with the donor qualities of and the steric hindrance at the pyridine/pyrimidine (pyr) feet, and was effected by two Npyr →ZnPor interactions. Accordingly, the sliders move over the three zinc porphyrins in the deck at different speeds, namely with 32.2, 220, and 440 kHz at room temperature. The addition of N-methylpyrrolidine as an organocatalyst to DS1-DS3 generates catalytic three-component machineries. By using a conjugate addition as a probe reaction, we observed a correlation between the operating speed of the slider-on-deck systems and the yields of the catalytic reaction. As the thermodynamic binding of the slider decreases, both the frequency of the sliding motion and the yield of the catalytic reaction increase.

A hydrophobic anchor mechanism defines a deacetylase family that suppresses host response against YopJ effectors

Several Pseudomonas and Xanthomonas species are plant pathogens that infect the model organism Arabidopsis thaliana and important crops such as Brassica. Resistant plants contain the infection by rapid cell death of the infected area through the hypersensitive response (HR). A family of highly related α/β hydrolases is involved in diverse processes in all domains of life. Functional details of their catalytic machinery, however, remained unclear. We report the crystal structures of α/β hydrolases representing two different clades of the family, including the protein SOBER1, which suppresses AvrBsT-incited HR in Arabidopsis. Our results reveal a unique hydrophobic anchor mechanism that defines a previously unknown family of protein deacetylases. Furthermore, this study identifies a lid-loop as general feature for substrate turnover in acyl-protein thioesterases and the described family of deacetylases. Furthermore, we found that SOBER1’s biological function is not restricted to Arabidopsis thaliana and not limited to suppress HR induced by AvrBsT.

Crystal structure of a marine glycoside hydrolase family 99-related protein lacking catalytic machinery.

Algal polysaccharides of diverse structures are one of the most abundant carbon resources for heterotrophic, marine bacteria with coevolved digestive enzymes. A putative sulfo-mannan polysaccharide utilization locus, which is conserved in marine flavobacteria, contains an unusual GH99-like protein that lacks the conserved catalytic residues of glycoside hydrolase family 99. Using X-ray crystallography, we structurally characterized this protein from the marine flavobacterium Ochrovirga pacifica to help elucidate its molecular function. The structure reveals the absence of potential catalytic residues for polysaccharide hydrolysis, which-together with additional structural features-suggests this protein may be noncatalytic and involved in carbohydrate binding.

Crystal Structure and Substrate Specificity Modification of Acetyl Xylan Esterase from Aspergillus luchuensis.

Acetyl xylan esterase (AXE) catalyzes the hydrolysis of the acetyl bonds present in plant cell wall polysaccharides. Here, we determined the crystal structure of AXE from Aspergillus luchuensis (AlAXEA), providing the three-dimensional structure of an enzyme in the Esterase_phb family. AlAXEA shares its core α/β-hydrolase fold structure with esterases in other families, but it has an extended central β-sheet at both its ends and an extra loop. Structural comparison with a ferulic acid esterase (FAE) from Aspergillus niger indicated that AlAXEA has a conserved catalytic machinery: a catalytic triad (Ser119, His259, and Asp202) and an oxyanion hole (Cys40 and Ser120). Near the catalytic triad of AlAXEA, two aromatic residues (Tyr39 and Trp160) form small pockets at both sides. Homology models of fungal FAEs in the same Esterase_phb family have wide pockets at the corresponding sites because they have residues with smaller side chains (Pro, Ser, and Gly). Mutants with site-directed mutations at Tyr39 showed a substrate specificity similar to that of the wild-type enzyme, whereas those with mutations at Trp160 acquired an expanded substrate specificity. Interestingly, the Trp160 mutants acquired weak but significant type B-like FAE activity. Moreover, the engineered enzymes exhibited ferulic acid-releasing activity from wheat arabinoxylan.IMPORTANCE Hemicelluloses in the plant cell wall are often decorated by acetyl and ferulic acid groups. Therefore, complete and efficient degradation of plant polysaccharides requires the enzymes for cleaving the side chains of the polymer. Since the Esterase_phb family contains a wide array of fungal FAEs and AXEs from fungi and bacteria, our study will provide a structural basis for the molecular mechanism of these industrially relevant enzymes in biopolymer degradation. The structure of the Esterase_phb family also provides information for bacterial polyhydroxyalkanoate depolymerases that are involved in biodegradation of thermoplastic polymers.

A DNA-Fueled and Catalytic Molecule Machine Lights Up Trace Under-Expressed MicroRNAs in Living Cells.

The detection of specific intracellular microRNAs (miRNAs) in living cells can potentially provide insight into the causal mechanism of cancer metastasis and invasion. However, because of the characteristic nature of miRNAs in terms of small sizes, low abundance, and similarity among family members, it is a great challenge to monitor miRNAs in living cells, especially those with much lower expression levels. In this work, we describe the establishment of a DNA-fueled and catalytic molecule machinery in cell signal amplification approach for monitoring trace and under-expressed miRNAs in living cells. The presence of the target miRNA releases the hairpin sequences from the dsDNA (containing the fluorescence resonance energy transfer (FRET) pair-labeled and unfolded hairpin sequences)-conjugated gold nanoparticles (dsDNA-AuNPs), and the DNA fuel strands assist the recycling of the target miRNA sequences via two cascaded strand displacement reactions, leading to the operation of the molecular machine in a catalytic fashion and the release of many hairpin sequences. As a result, the liberated hairpin sequences restore the folded hairpin structures and bring the FRET pair into close proximity to generate significantly amplified signals for detecting trace miRNA targets. Besides, the dsDNA-AuNP nanoprobes have good nuclease stability and show low cytotoxicity to cells, and the application of such a molecular system for monitoring trace and under-expressed miRNAs in living cells has also been demonstrated. With the advantages of in cell signal amplification and reduced background noise, the developed method thus offers new opportunities for detecting various trace intracellular miRNA species.

Optimal management of nutrient reserves in microorganisms under time-varying environmental conditions

Intracellular reserves are a conspicuous feature of many bacteria; such internal stores are often present in the form of inclusions in which polymeric storage compounds are accumulated. Such reserves tend to increase in times of plenty and be used up in times of scarcity. Mathematical models that describe the dynamical nature of reserve build-up and use are known as “cell quota,” “dynamic energy/nutrient budget,” or “variable-internal-stores” models. Here we present a stoichiometrically consistent macro-chemical model that accounts for variable stores as well as adaptive allocation of building blocks to various types of catalytic machinery. The model posits feedback loops linking expression of assimilatory machinery to reserve density. The precise form of the “regulatory law” at the heart of such a loop expresses how the cell manages internal stores. We demonstrate how this “regulatory law” can be recovered from experimental data using several empirical data sets. We find that stores should be expected to be negligibly small in stable growth-sustaining environments, but prominent in environments characterised by marked fluctuations on time scales commensurate with the inherent dynamic time scale of the organismal system.

Target Residence Time-Guided Optimization on TTK Kinase Results in Inhibitors with Potent Anti-Proliferative Activity

The protein kinase threonine tyrosine kinase (TTK; also known as Mps1) is a critical component of the spindle assembly checkpoint and a promising drug target for the treatment of aggressive cancers, such as triple negative breast cancer. While the first TTK inhibitors have entered clinical trials, little is known about how the inhibition of TTK with small-molecule compounds affects cellular activity. We studied the selective TTK inhibitor NTRC 0066-0, which was developed in our own laboratory, together with 11 TTK inhibitors developed by other companies, including Mps-BAY2b, BAY 1161909, BAY 1217389 (Bayer), TC-Mps1-12 (Shionogi), and MPI-0479605 (Myrexis). Parallel testing shows that the cellular activity of these TTK inhibitors correlates with their binding affinity to TTK and, more strongly, with target residence time. TTK inhibitors are therefore an example where target residence time determines activity in in vitro cellular assays. X-ray structures and thermal stability experiments reveal that the most potent compounds induce a shift of the glycine-rich loop as a result of binding to the catalytic lysine at position 553. This “lysine trap” disrupts the catalytic machinery. Based on these insights, we developed TTK inhibitors, based on a (5,6-dihydro)pyrimido[4,5-e]indolizine scaffold, with longer target residence times, which further exploit an allosteric pocket surrounding Lys553. Their binding mode is new for kinase inhibitors and can be classified as hybrid Type I/Type III. These inhibitors have very potent anti-proliferative activity that rivals classic cytotoxic therapy. Our findings will open up new avenues for more applications for TTK inhibitors in cancer treatment.

Insight into the remarkable affinity and selectivity of the aminobenzosuberone scaffold for the M1 aminopeptidases family based on structure analysis.

Aminopeptidases are ubiquitous hydrolases that cleave the N-terminal residues of proteins and oligopeptides. They are broadly distributed throughout all kingdoms of life and have been implicated in a wide variety of physiological processes, including viral infection, parasite metabolism, protein processing, regulation of peptide hormones, and cancer cell proliferation. Members of the M1 family, also termed gluzincins, are defined by two highly conserved motifs in the catalytic domain: a zinc-binding motif, HEXXH-(X18)-E; and an exopeptidase motif, GXMEN. We report the high-resolution X-ray structures of E. coli aminopeptidase N (PepN) in complex with three aminobenzosuberone scaffolds that display various Ki values (50, 0.33, and 0.034 µM) and provide a compelling view of the outstanding selectivity of these chemical entities for the M1 aminopeptidases. This series of inhibitors interacts as transition state mimics with highly conserved residues of the catalytic machinery and substrate recognition sites. Structural comparisons and model-building studies allowed a deep interpretation of the SAR observed for bacterial, as well as mammalian enzymes. Proteins 2017; 85:1413-1421. © 2017 Wiley Periodicals, Inc.

MFN1 structures reveal nucleotide-triggered dimerization critical for mitochondrial fusion.

Mitochondria are double-membrane organelles with varying shapes influenced by metabolic conditions, developmental stage, and environmental stimuli1–4. Their dynamic morphology is realized through regulated and balanced fusion and fission processes5, 6. Fusion is crucial for the health and physiological functions of mitochondria, including complementation of damaged mitochondrial DNAs and maintenance of membrane potential6–8. Mitofusins (Mfns) are dynamin-related GTPases essential for mitochondrial fusion9, 10. They are embedded in the mitochondrial outer membrane and thought to fuse adjacent mitochondria via concerted oligomerization and GTP hydrolysis11–13. However, the molecular mechanisms behind this process remains elusive. Here we present crystal structures of engineered human Mfn1 containing the GTPase domain and a helical domain in different stages of GTP hydrolysis. The helical domain is composed of elements from widely dispersed sequence regions of Mfn1 and resembles the Neck of the bacterial dynamin-like protein. The structures reveal unique features of its catalytic machinery and explain how GTP binding induces conformational changes to promote G domain dimerization in the transition state. Disruption of G domain dimerization abolishes the fusogenic activity of Mfn1. Moreover, a conserved aspartate trigger was found in Mfn1 to affect mitochondrial elongation, likely through a GTP-loading-dependent domain rearrangement. Based on these results, we propose a mechanistic model for Mfn1-mediated mitochondrial tethering. Our study provides important insights in the molecular basis of mitochondrial fusion and mitofusin-related human neuromuscular disorders14.

Recognition of protein-linked glycans as a determinant of peptidase activity

The vast majority of proteins are posttranslationally altered, with the addition of covalently linked sugars (glycosylation) being one of the most abundant modifications. However, despite the hydrolysis of protein peptide bonds by peptidases being a process essential to all life on Earth, the fundamental details of how peptidases accommodate posttranslational modifications, including glycosylation, has not been addressed. Through biochemical analyses and X-ray crystallographic structures we show that to hydrolyze their substrates, three structurally related metallopeptidases require the specific recognition of O-linked glycan modifications via carbohydrate-specific subsites immediately adjacent to their peptidase catalytic machinery. The three peptidases showed selectivity for different glycans, revealing protein-specific adaptations to particular glycan modifications, yet always cleaved the peptide bond immediately preceding the glycosylated residue. This insight builds upon the paradigm of how peptidases recognize substrates and provides a molecular understanding of glycoprotein degradation.

Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism.

The amount of cholesterol made by many plants is not negligible. Whereas cholesterogenesis in animals was elucidated decades ago, the plant pathway has remained enigmatic. Among other roles, cholesterol is a key precursor for thousands of bioactive plant metabolites, including the well-known Solanum steroidal glycoalkaloids. Integrating tomato transcript and protein co-expression data revealed candidate genes putatively associated with cholesterol biosynthesis. A combination of functional assays including gene silencing, examination of recombinant enzyme activity and yeast mutant complementation suggests the cholesterol pathway comprises 12 enzymes acting in 10 steps. It appears that half of the cholesterogenesis-specific enzymes evolved through gene duplication and divergence from phytosterol biosynthetic enzymes, whereas others act reciprocally in both cholesterol and phytosterol metabolism. Our findings provide a unique example of nature's capacity to exploit existing protein folds and catalytic machineries from primary metabolism to assemble a new, multi-step metabolic pathway. Finally, the engineering of a 'high-cholesterol' model plant underscores the future value of our gene toolbox to produce high-value steroidal compounds via synthetic biology.

Mannoside recognition and degradation by bacteria.

Mannosides constitute a vast group of glycans widely distributed in nature. Produced by almost all organisms, these carbohydrates are involved in numerous cellular processes, such as cell structuration, protein maturation and signalling, mediation of protein-protein interactions and cell recognition. The ubiquitous presence of mannosides in the environment means they are a reliable source of carbon and energy for bacteria, which have developed complex strategies to harvest them. This review focuses on the various mannosides that can be found in nature and details their structure. It underlines their involvement in cellular interactions and finally describes the latest discoveries regarding the catalytic machinery and metabolic pathways that bacteria have developed to metabolize them.

Structural Insight into the Enzymatic Formation of Bacterial Stilbene

In contrast to stilbene biosynthesis by type III polyketide synthase in plants, in bacteria stilbene is produced by the collaboration of two enzymes in Photorhabdus luminescens: the unusual β-ketosynthase StlD catalyzes the condensation of the β-ketoacyl starter with an α,β-unsaturated-acyl substrate (two C-C bond-forming reactions) to produce isopropylstyrylcyclohexanedione, which is subsequently converted to stilbene by the aromatase StlC. Here we report the invitro characterizations of StlD and StlC, and the X-ray crystal structures of StlD. Interestingly, structure-based mutagenesis demonstrated that His302, within the conserved Cys-His-Asn triad, is not essential for the enzyme reaction, while Glu154 functions as a base-catalyst to activate the β-ketoacyl intermediate bound to the catalytic Cys126. The structures also revealed the presence of a putative nucleophilic water molecule activated by hydrogen bond networks with Glu154 and Ser340, suggesting that StlD employs novel catalytic machinery for the condensation of two acyl substrates to produce the cyclohexanedione scaffold.

N-Linked glycans are an important component of the processive machinery of cellobiohydrolases

Cellobiohydrolases (CBHs), belonging to glycoside hydrolase families 6 and 7 (GH6 and GH7), are the major components of cellulase systems of filamentous fungi involved in biodegradation of cellulose in nature. Previous studies demonstrated that N-linked glycans in the catalytic domains of GH7 CBHs significantly affect the enzyme activity against cellulosic substrates. The influence of N-linked glycans on the activity and processivity of recombinant GH6 CBH II from Penicillium verruculosum (PvCel6A) was studied using site-directed mutagenesis of the respective Asn residues. Depending on the position of N-glycans on the surface of a protein globule, they affected the enzyme activity against cellulose either negatively or positively. The decrease or increase in the degree of processivity of recombinant forms of PvCel6A generally correlated with activity changes against Avicel. The mechanism of the N-glycan influence seems to be universal for GH6 and GH7 CBHs. The observed effects for CBHs from both families are explained in terms of a mechanistic model that also makes clear our previously published data on the highly active CBH IIb from Myceliophthora thermophila (MtCel6B). This study, together with data of other researchers, strongly suggests that the N-linked glycans in the catalytic domains of GH6 and GH7 CBHs are involved in processive catalytic machinery of these enzymes. Data obtained should be taken into account during development of new and more effective biocatalysts by protein engineering techniques.

Photoreduction of Shewanella oneidensis Extracellular Cytochromes by Organic Chromophores and Dye-Sensitized TiO2.

The transfer of photoenergized electrons from extracellular photosensitizers across a bacterial cell envelope to drive intracellular chemical transformations represents an attractive way to harness nature's catalytic machinery for solar‐assisted chemical synthesis. In Shewanella oneidensis MR‐1 (MR‐1), trans‐outer‐membrane electron transfer is performed by the extracellular cytochromes MtrC and OmcA acting together with the outer‐membrane‐spanning porin⋅cytochrome complex (MtrAB). Here we demonstrate photoreduction of solutions of MtrC, OmcA, and the MtrCAB complex by soluble photosensitizers: namely, eosin Y, fluorescein, proflavine, flavin, and adenine dinucleotide, as well as by riboflavin and flavin mononucleotide, two compounds secreted by MR‐1. We show photoreduction of MtrC and OmcA adsorbed on RuII‐dye‐sensitized TiO2 nanoparticles and that these protein‐coated particles perform photocatalytic reduction of solutions of MtrC, OmcA, and MtrCAB. These findings provide a framework for informed development of strategies for using the outer‐membrane‐associated cytochromes of MR‐1 for solar‐driven microbial synthesis in natural and engineered bacteria.