Catalytic Histidine Research Articles

Understanding stevia’s sweetness

Researchers have crystallized the enzyme that makes the natural product stevia taste 200 times as sweet as sugar. The enzyme is a uridine diphosphate–dependent glucosyltransferase, UGT76G1, and it catalyzes the addition of branched glucosides to compounds in stevia—primarily two diterpenoids called stevioside and rebaudioside A (Proc. Natl. Acad. Sci. U.S.A. 2019, DOI: 10.1073/pnas.1902104116). Without the branched glucosides, says lead researcher Joseph Jez of Washington University in St. Louis, stevia loses its sweetness. To get the crystal structure, Jez’s team bound rebaudioside A to UGT76G1, revealing a catalytic histidine as well as a large cavity that accommodates the growing branched chain. Jez says that the large cavity is unique to this glycosyltransferase: others found in stevia plants seem to add glucosides more linearly. Richard Dixon, a plant biochemist at the University of North Texas who was not involved in the research, says that knowing this structure will help food scientists create

Molecular basis for branched steviol glucoside biosynthesis

Steviol glucosides, such as stevioside and rebaudioside A, are natural products roughly 200-fold sweeter than sugar and are used as natural, noncaloric sweeteners. Biosynthesis of rebaudioside A, and other related stevia glucosides, involves formation of the steviol diterpenoid followed by a series of glycosylations catalyzed by uridine diphosphate (UDP)-dependent glucosyltransferases. UGT76G1 from Stevia rebaudiana catalyzes the formation of the branched-chain glucoside that defines the stevia molecule and is critical for its high-intensity sweetness. Here, we report the 3D structure of the UDP-glucosyltransferase UGT76G1, including a complex of the protein with UDP and rebaudioside A bound in the active site. The X-ray crystal structure and biochemical analysis of site-directed mutants identifies a catalytic histidine and how the acceptor site of UGT76G1 achieves regioselectivity for branched-glucoside synthesis. The active site accommodates a two-glucosyl side chain and provides a site for addition of a third sugar molecule to the C3' position of the first C13 sugar group of stevioside. This structure provides insight on the glycosylation of other naturally occurring sweeteners, such as the mogrosides from monk fruit, and a possible template for engineering of steviol biosynthesis.

Nonnatural amino acid amps up enzyme efficiency

The combination of protein design and directed evolution is a powerful way to create enzymes with new functions. Now, Anthony P. Green and coworkers at the University of Manchester have thrown a nonnatural amino acid into the mix. They started with a protein computationally designed to catalyze Morita-Baylis-Hillman carbon-carbon bond formation and transformed it into an enzyme that catalyzes ester hydrolysis (Nature 2019, DOI: 10.1038/s41586-019-1262-8). First, they replaced the catalytic histidine in the active site with a noncanonical methylhistidine. Then they subjected that new enzyme to multiple rounds of laboratory evolution with an assay based on fluorescein detection. The most active variant hydrolyzed fluorescein 2-phenylacetate 9,000 times as efficiently as free methylhistidine in solution and 2,800 times as efficiently as the organocatalysts dimethylaminopyridine and N-methyl imidazole. Further rounds of evolution gave rise to an enantioselective enzyme that preferentially hydrolyzes one of th...

GII.4 Norovirus Protease Shows pH-Sensitive Proteolysis with a Unique Arg-His Pairing in the Catalytic Site.

Human noroviruses (NoVs) are the main cause of epidemic and sporadic gastroenteritis. Phylogenetically, noroviruses are divided into seven genogroups, with each divided into multiple genotypes. NoVs belonging to genogroup II and genotype 4 (GII.4) are globally most prevalent. Genetic diversity among the NoVs and the periodic emergence of novel strains present a challenge for the development of vaccines and antivirals to treat NoV infection. NoV protease is essential for viral replication and is an attractive target for the development of antivirals. The available structure of GI.1 protease provided a basis for the design of inhibitors targeting the active site of the protease. These inhibitors, although potent against the GI proteases, poorly inhibit the GII proteases, for which structural information is lacking. To elucidate the structural basis for this difference in the inhibitor efficiency, we determined the crystal structure of a GII.4 protease. The structure revealed significant changes in the S2 substrate-binding pocket, making it noticeably smaller, and in the active site, with the catalytic triad residues showing conformational changes. Furthermore, a conserved arginine is found inserted into the active site, interacting with the catalytic histidine and restricting substrate/inhibitor access to the S2 pocket. This interaction alters the relationships between the catalytic residues and may allow for a pH-dependent regulation of protease activity. The changes we observed in the GII.4 protease structure may explain the reduced potency of the GI-specific inhibitors against the GII protease and therefore must be taken into account when designing broadly cross-reactive antivirals against NoVs.IMPORTANCE Human noroviruses (NoVs) cause sporadic and epidemic gastroenteritis worldwide. They are divided into seven genogroups (GI to GVII), with each genogroup further divided into several genotypes. Human NoVs belonging to genogroup II and genotype 4 (GII.4) are the most prevalent. Currently, there are no vaccines or antiviral drugs available for NoV infection. The protease encoded by NoV is considered a valuable target because of its essential role in replication. NoV protease structures have only been determined for the GI genogroup. We show here that the structure of the GII.4 protease exhibits several significant changes from GI proteases, including a unique pairing of an arginine with the catalytic histidine that makes the proteolytic activity of GII.4 protease pH sensitive. A comparative analysis of NoV protease structures may provide a rational framework for structure-based drug design of broadly cross-reactive inhibitors targeting NoVs.

A Novel Phospholipase A2 Isolated from Palythoa caribaeorum Possesses Neurotoxic Activity.

Zoanthids of the genus Palythoa are distributed worldwide in shallow waters around coral reefs. Like all cnidarians, they possess nematocysts that contain a large diversity of toxins that paralyze their prey. This work was aimed at isolating and functionally characterizing a cnidarian neurotoxic phospholipase named A2-PLTX-Pcb1a for the first time. This phospholipase was isolated from the venomous extract of the zoanthid Palythoa caribaeorum. This enzyme, which is Ca2+-dependent, is a 149 amino acid residue protein. The analysis of the A2-PLTX-Pcb1a sequence showed neurotoxic domain similitude with other neurotoxic sPLA2´s, but a different catalytic histidine domain. This is remarkable, since A2-PLTX-Pcb1a displays properties like those of other known PLA2 enzymes.

Kinetic model of the enzymatic Michael addition for synthesis of mitomycin analogs catalyzed by immobilized lipase from T. laibacchii

The present study investigates the kinetic model of the enzymatic Michael addition of butylamine to 2-methyl-1,4-benzoquinone to form 2-methyl-3-n-butylaminoyl-1-hydro-4-quinone in citrate buffer solution (pH 7.0). The yield of the product of 98% was achieved, mainly due to the excellent regioselectivity of immobilized lipase from T. laibacchii. The immobilized preparation used here was obtained by a method of purification and in situ immobilization. Through the purification using a PEG 4000/ K2HPO4 aqueous two-phase system (ATPS), the T. laibacchii lipase was partitioned predominantly in the PEG-rich top phase where diatomite was added to achieve in situ immobilization via interfacial activation on the hydrophobic support. A proposed reaction mechanism of the Michael addition involves (1) the oxyanion hole polarizes the α,β-unsaturated carbonyl of 2-methyl-1,4 -benzoquinone, increasing its electrophilic ability, (2) the catalytic histidine deprotonates the nucleophile n-butyl amine. A modified sequential mechanism including ordered and random sequential bi-bi was proposed for the first, and it is beneficial to add these modification mechanisms to the family of enzyme complex reaction mechanism because the mechanism is partly expanded. The kinetic parameters were directly obtained by combining the numerical integration toolbox ode45 to solve differential equations and the nonlinear optimization toolbox fmincon for error minimizing objective function. A very satisfactory agreement between experimental data and model results was obtained based on the modified random bi-bi mechanism, implying that the enzymatic Michael addition may follow the modified random bi-bi mechanism. The mass transfer limitations were investigated, and it is found that both internal and external mass transfer limitations could be ignored.

Targeting Tyrosyl-DNA phosphodiesterase I to enhance toxicity of phosphodiester linked DNA-adducts.

Our genomic DNA is under constant assault from endogenous and exogenous sources, which needs to be resolved to maintain cellular homeostasis. The eukaryotic DNA repair enzyme Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the hydrolysis of phosphodiester bonds that covalently link adducts to DNA-ends. Tdp1 utilizes two catalytic histidines to resolve a growing list of DNA-adducts. These DNA-adducts can be divided into two groups: small adducts, including oxidized nucleotides, RNA, and non-canonical nucleoside analogs, and large adducts, such as (drug-stabilized) topoisomerase- DNA covalent complexes or failed Schiff base reactions as occur between PARP1 and DNA. Many Tdp1 substrates are generated by chemotherapeutics linking Tdp1 to cancer drug resistance, making a compelling argument to develop small molecules that target Tdp1 as potential novel therapeutic agents. Tdp1’s unique catalytic cycle, which is centered on the formation of Tdp1-DNA covalent reaction intermediate, allows for two principally different targeting strategies: (1) catalytic inhibition of Tdp1 catalysis to prevent Tdp1-mediated repair of DNA-adducts that enhances the effectivity of chemotherapeutics; and (2) poisoning of Tdp1 by stabilization of the Tdp1- DNA covalent reaction intermediate, which would increase the half-life of a potentially toxic DNA-adduct by preventing its resolution, analogous to topoisomerase targeted poisons such as topotecan or etoposide. The catalytic Tdp1 mutant that forms the molecular basis of the autosomal recessive neurodegenerative disease spinocerebellar ataxia with axonal neuropathy best illustrates this concept; however, no small molecules have been reported for this strategy. Herein, we concisely discuss the development of Tdp1 catalytic inhibitors and their results.

Distinctive structural motifs co-ordinate the catalytic nucleophile and the residues of the oxyanion hole in the alpha/beta-hydrolase fold enzymes.

The alpha/beta-hydrolases (ABH) are among the largest structural families of proteins that are found in nature. Although they vary in their sequence and function, the ABH enzymes use a similar acid-base-nucleophile catalytic mechanism to catalyze reactions on different substrates. Because ABH enzymes are biocatalysts with a wide range of potential applications, protein engineering has taken advantage of their catalytic versatility to develop enzymes with industrial applications. This study is a comprehensive analysis of 40 ABH enzyme families focusing on two identified substructures: the nucleophile zone and the oxyanion zone, which co-ordinate the catalytic nucleophile and the residues of the oxyanion hole, and independently reported as critical for the enzymatic activity. We also frequently observed an aromatic cluster near the nucleophile and oxyanion zones, and opposite the ligand-binding site. The nucleophile zone, the oxyanion zone and the residue cluster enriched in aromatic side chains comprise a three-dimensional structural organization that shapes the active site of ABH enzymes and plays an important role in the enzymatic function by structurally stabilizing the catalytic nucleophile and the residues of the oxyanion hole. The structural data support the notion that the aromatic cluster can participate in co-ordination of the catalytic histidine loop, and properly place the catalytic histidine next to the catalytic nucleophile.

The structure of the deubiquitinase USP15 reveals a misaligned catalytic triad and an open ubiquitin-binding channel

Ubiquitin-specific protease 15 (USP15) regulates important cellular processes, including transforming growth factor β (TGF-β) signaling, mitophagy, mRNA processing, and innate immune responses; however, structural information on USP15's catalytic domain is currently unavailable. Here, we determined crystal structures of the USP15 catalytic core domain, revealing a canonical USP fold, including a finger, palm, and thumb region. Unlike for the structure of paralog USP4, the catalytic triad is in an inactive configuration with the catalytic cysteine ∼10 Å apart from the catalytic histidine. This conformation is atypical, and a similar misaligned catalytic triad has so far been observed only for USP7, although USP15 and USP7 are differently regulated. Moreover, we found that the active-site loops are flexible, resulting in a largely open ubiquitin tail–binding channel. Comparison of the USP15 and USP4 structures points to a possible activation mechanism. Sequence differences between these two USPs mainly map to the S1′ region likely to confer specificity, whereas the S1 ubiquitin–binding pocket is highly conserved. Isothermal titration calorimetry monoubiquitin- and linear diubiquitin-binding experiments showed significant differences in their thermodynamic profiles, with USP15 displaying a lower affinity for monoubiquitin than USP4. Moreover, we report that USP15 is weakly inhibited by the antineoplastic agent mitoxantrone in vitro. A USP15–mitoxantrone complex structure disclosed that the anthracenedione interacts with the S1′ binding site. Our results reveal first insights into USP15's catalytic domain structure, conformational changes, differences between paralogs, and small-molecule interactions and establish a framework for cellular probe and inhibitor development.

84 - Nitro fatty acids as activators of hSIRT6 deacetylase activity

Sirtuins are NAD+-dependent deacetylases reported as key factors in metabolism, regulation, inflammation and stress response. Human sirtuin 6 (hSIRT6) has been shown in vivo to efficiently deacetylase histone 3, however, in vitro displays low deacetylase activity and possess long-chain fatty acid deacylase activity. So far, only a few compounds have been identified to stimulate the deacetylation activity of SIRT6: long-chain free fatty acids and some polyphenols, specifically quercetin and luteolin, at high concentrations. In this study, recombinant hSIRT6 was expressed and purified from E. coli and zinc content/mole protein was determined. Deacylase activity was measured either using a coupled-enzyme assay (plus nicotinamidase and glutamate dehydrogenase under non-limiting conditions) following NADPH consumption at 340 nm, or by quantifying deacylated peptide product by hplc with time. Synthetic acetylated and myristoylated histone 3 peptides containing tryptophans (H3K9-Ac, H3K9-Myr) were used as substrates. In vitro, hSIRT6 displayed better demyristoilase activity than deacetylase activity; Km for H3K9-Ac > 400 μM while Km = 18 μM for H3K9-Myr. The increase in basal deacetylase activity in the presence of μM oleic acid was confirmed. Surprisingly, treatment with the electrophilic derivative nitro-oleic acid do not inactivate, on the contrary, enhanced deacetylase activity, albeit the presence of a catalytic histidine. The formation of a Michael adduct with cysteine residues was confirmed. In addition, tertiary structure changes after incubation with the nitroalkene was evidenced by near-UV CD spectra, that is likely responsible for the observed reduction in Km for the acetylated peptide substrate. Nitro oleic acid had no significant effect on the demyristoilase activity, thus appears as an specific activator of hSIRT6 deacetylase activity.

X-ray structures of human bile-salt activated lipase conjugated to nerve agents surrogates

The efficiency of human butyrylcholinesterase (BChE) as a stoichiometric bioscavenger of nerve agents is well established. However, wide use is currently limited by production and purification costs. Aiming at identifying an alternative human protein bioscavenger, we looked for an original scaffold candidate by virtual screening of the Protein Data Bank for functional similarity using the "Surfing the Molecules" software (sumo-pbil.ibcp.fr) and a search model based on the BChE active site topology. Besides the expected acetylcholinesterase and butyrylcholinesterase, we identified a set of bile salt activated lipases structures, among which the human pancreatic lipase (hBAL) that shares 34% identity with BChE. We produced the recombinant enzyme in mammalian cells, purified it, and measured the inhibition constants for paraoxon and surrogates of VX, sarin and tabun. We solved the X-ray structure of apo hBAL and conjugates with paraoxon and the surrogates at resolutions in the 2-Å range. These structures allow the assessment of hBAL for scavenging nerve agents. They revealed that hBAL has inverted stereoselectivity for the surrogates of nerve agent compared to human cholinesterases. We observed a remarkable flip of the catalytic histidine driven by the chelation of Zn2+. Dealkylation of the conjugate, aka aging, was solely observed for paraoxon.

Crystal structure of a membrane-bound O-acyltransferase.

Membrane-bound O-acyltransferases (MBOATs) represent a superfamily of integral transmembrane enzymes found in all kingdoms of life1. In bacteria, MBOATs modify protective cell surface polymers. In vertebrates, some MBOAT enzymes such as acyl-CoA:cholesterol acyltransferase (ACAT) and diacylglycerol acyltransferase 1 (DGAT1) are responsible for lipid biosynthesis and phospholipid remodeling2,3. Some other MBOATs, including porcupine (PORCN), hedgehog acyltransferase (HHAT) and ghrelin acyltransferase (GOAT), catalyze essential lipid modifications of secreted proteins such as Wnt, hedgehog and ghrelin, respectively4–10. Although many MBOAT proteins are important drug targets, little is known about their molecular architecture and functional mechanisms. Here we present crystal structures of DltB, a MBOAT responsible for D-alanylation of cell wall teichoic acid (TA) of Gram-positive bacteria11–16, by itself and in complex with the D-alanyl donor protein, DltC. DltB contains a ring of 11 peripheral transmembrane helices, which shield a highly conserved extracellular structural “funnel” extending into the middle of lipid bilayer. The conserved catalytic histidine residue is located at the bottom of this funnel and connected to the intracellular DltC through a narrow tunnel. Mutation of either the catalytic histidine or the DltC binding site of DltB abolishes LTA D-alanylation, and sensitizes the Gram-positive bacterium Bacillus subtilis to cell wall stress, suggesting cross-membrane catalysis involving the tunnel. Structure-guided sequence comparison among DltB and vertebrate MBOATs reveals a conserved structural core and suggests similar catalytic mechanisms. Our structures provide a template for understanding MBOAT structure-function relationships and for developing therapeutic MBOAT inhibitors.

Structure of the bifunctional cryptochrome aCRY from Chlamydomonas reinhardtii.

Photolyases and cryptochromes form an almost ubiquitous family of blue light photoreceptors involved in the repair and maintenance of DNA integrity or regulatory control. We found that one cryptochrome from the green alga Chlamydomonas reinhardtii (CraCRY) is capable of both, control of transcript levels and the sexual cycle of the alga in a positive (germination) and negative manner (mating ability), as well as catalyzing the repair of UV-DNA lesions. Its 1.6 Å crystal structure shows besides the FAD chromophore an aromatic tetrad that is indispensable in animal-like type I cryptochromes for light-driven change of their signaling-active redox state and formation of a stable radical pair. Given CraCRY’s catalytic activity as (6-4) photolyase in vivo and in vitro, we present the first co-crystal structure of a cryptochrome with duplex DNA comprising a (6-4) pyrimidine–pyrimidone lesion. This 2.9 Å structure reveals a distinct conformation for the catalytic histidine His1, H357, that challenges previous models of a single-photon driven (6-4) photolyase mechanism.

Enhanced catalytic activities and modified substrate preferences for taxoid 10β-O-acetyl transferase mutants by engineering catalytic histidine residues

Taxoid 10β-O-acetyl transferase (DBAT) was redesigned to enhance its catalytic activity and substrate preference for baccatin III and taxol biosynthesis. Residues H162, D166 and R363 were determined as potential sites within the catalytic pocket of DBAT for molecular docking and site-directed mutagenesis to modify the activity of DBAT. Enzymatic activity assays revealed that the kcat/KM values of mutant H162A/R363H, D166H, R363H, D166H/R363H acting on 10-deacetylbaccatin III were about 3, 15, 26 and 60 times higher than that of the wild type of DBAT, respectively. Substrate preference assays indicated that these mutants (H162A/R363H, D166H, R363H, D166H/R363H) could transfer acetyl group from unnatural acetyl donor (e.g. vinyl acetate, sec-butyl acetate, isobutyl acetate, amyl acetate and isoamyl acetate) to 10-deacetylbaccatin III. Taxoid 10β-O-acetyl transferase mutants with redesigned active sites displayed increased catalytic activities and modified substrate preferences, indicating their possible application in the enzymatic synthesis of baccatin III and taxol.

Insights into catalysis and function of phosphoribosyl-linked serine ubiquitination.

Conventional ubiquitination regulates key cellular processes by catalyzing the ATP-dependent formation of an isopeptide bond between ubiquitin (Ub) and primary amines in substrate proteins1. Recently, SidE family of bacterial effector proteins (SdeA, SdeB, SdeC and SidE) of pathogenic Legionella pneumophila were shown to utilize NAD+ to mediate phosphoribosyl-linked ubiquitination (PR-ubiquitination) of serine residues in host proteins2,3. Yet, the molecular architecture of the catalytic platform enabling such a complex multistep process remained unknown. Here, we describe the structure of the catalytic core of SdeA composed of the mono-ADP-ribosyltransferase (mART) and the phosphodiesterase (PDE) domains and shed light on the activity of two distinct catalytic sites for serine ubiquitination. The mART catalytic site is composed of an α-helical lobe (AHL) that together with the mART-core creates a chamber for NAD+ binding and ADP-ribosylation of Ub. The catalytic site in the PDE domain cleaves ADP-ribosylated Ub to phosphoribosyl Ub (PR-Ub) and mediates a two-step PR-Ub transfer reaction: first to a catalytic histidine 277 (forming a transient SdeA:H277-PR-Ub intermediate) and subsequently to a serine residue in host proteins. Structural analysis revealed a substrate binding cleft in the PDE domain juxtaposing the catalytic site that is essential for serine positioning for ubiquitination. Using degenerate substrate peptides and newly identified ubiquitination sites in RTN4B, we show that disordered polypeptides with hydrophobic residues surrounding the target serine residues are preferred substrates for SdeA ubiquitination. Infection studies with L. pneumophila expressing substrate-binding mutants of SdeA revealed that substrate ubiquitination rather than modification of the cellular Ub pool determines the pathophysiological effect of SdeA during acute bacterial infection.

Evolution of Caspase Allostery and Enzyme Specificity

We used phylogenetic, structural, and biophysical studies to examine the evolution of enzyme specificity and allosteric regulation in caspases. Caspase‐3 activation and function has been well defined during programmed cell death, but caspase activity, at low levels, is also required for cell development. We established a database, the CaspBase, for phylogenetic and ancestral reconstruction of caspases, which consists of >1,500 caspase genes. From this database, we resurrected ancestors of the caspase‐3, ‐6, ‐7 subfamily, and the common ancestor displays enzyme specificity similar to the initiator caspase subfamily from which it evolved. The specificities of the caspase‐3/‐7 and caspase‐6 enzymes evolved early, after the two lineages split. Structural studies of the ancestral enzymes identify changes in the active sites that result in changes in substrate selection. In contrast to enzyme activity, several allosteric sites evolved later in the lineages. We define interaction networks that facilitate the allosteric regulation in caspase‐3, and we show that, within a conserved loop, one site of phosphorylation evolved with the apoptotic caspases, while a second site is a more recent evolutionary event in mammalian caspase‐3. Localized changes in the loop propagate to the active site of the same protomer and disrupt substrate hydrolysis by facilitating transient ionic interactions with the catalytic histidine. In contrast, a cluster of hydrophobic amino acids in the dimer interface connects the conserved loop to the active site of the second protomer. The presence of the second modification in the conserved loop introduces a “kill switch” in mammalian caspase‐3. The data reveal how evolutionary changes in a conserved allosteric site result in both a common pathway for lowering activity during cell development as well as introducing a more recent cluster‐specific switch to abolish activity.Support or Funding InformationUT ArlingtonThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural Insights into the Free-Standing Condensation Enzyme SgcC5 Catalyzing Ester-Bond Formation in the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027

C-1027 is a chromoprotein enediyne antitumor antibiotic, consisting of the CagA apoprotein and the C-1027 chromophore. The C-1027 chromophore features a nine-membered enediyne core appended with three peripheral moieties, including an ( S)-3-chloro-5-hydroxy-β-tyrosine. In a convergent biosynthesis of the C-1027 chromophore, the ( S)-3-chloro-5-hydroxy-β-tyrosine moiety is appended to the enediyne core by the free-standing condensation enzyme SgcC5. Unlike canonical condensation domains from the modular nonribosomal peptide synthetases that catalyze amide-bond formation, SgcC5 catalyzes ester-bond formation, as demonstrated in vitro, between SgcC2-tethered ( S)-3-chloro-5-hydroxy-β-tyrosine and ( R)-1-phenyl-1,2-ethanediol, a mimic of the enediyne core as an acceptor substrate. Here, we report that (i) genes encoding SgcC5 homologues are widespread among both experimentally confirmed and bioinformatically predicted enediyne biosynthetic gene clusters, forming a new clade of condensation enzymes, (ii) SgcC5 shares a similar overall structure with the canonical condensation domains but forms a homodimer in solution, the active site of which is located in a cavity rather than a tunnel typically seen in condensation domains, and (iii) the catalytic histidine of SgcC5 activates the 2-hydroxyl group, while a hydrogen-bond network in SgcC5 prefers the R-enantiomer of the acceptor substrate, accounting for the regio- and stereospecific ester-bond formation between SgcC2-tethered ( S)-3-chloro-5-hydroxy-β-tyrosine and ( R)-1-phenyl-1,2-ethanediol upon acid-base catalysis. These findings expand the catalytic repertoire and reveal new insights into the structure and mechanism of condensation enzymes.

Catalytic Mechanism of Cruzain from Trypanosoma cruzi As Determined from Solvent Kinetic Isotope Effects of Steady-State and Pre-Steady-State Kinetics.

Cruzain, an important drug target for Chagas disease, is a member of clan CA of the cysteine proteases. Understanding the catalytic mechanism of cruzain is vital to the design of new inhibitors. To this end, we have determined pH-rate profiles for substrates and affinity agents and solvent kinetic isotope effects in pre-steady-state and steady-state modes using three substrates: Cbz-Phe-Arg-AMC, Cbz-Arg-Arg-AMC, and Cbz-Arg-Ala-AMC. The pH-rate profile of kcat/ Km for Cbz-Arg-Arg-AMC indicated p K1 = 6.6 (unprotonated) and p K2 ∼ 9.6 (protonated) groups were required for catalysis. The temperature dependence of the p K = 6.2-6.6 group exhibited a Δ Hion value of 8.4 kcal/mol, typical of histidine. The pH-rate profile of inactivation by iodoacetamide confirmed that the catalytic cysteine possesses a p Ka of 9.8. Normal solvent kinetic isotope effects were observed for both D2O kcat (1.6-2.1) and D2O kcat/ Km (1.1-1.4) for all three substrates. Pre-steady-state kinetics revealed exponential bursts of AMC production for Cbz-Phe-Arg-AMC and Cbz-Arg-Arg-AMC, but not for Cbz-Arg-Ala-AMC. The overall solvent isotope effect on kcat can be attributed to the solvent isotope effect on the deacylation step. Our results suggest that cruzain is unique among papain-like cysteine proteases in that the catalytic cysteine and histidine have neutral charges in the free enzyme. The generation of the active thiolate of the catalytic cysteine is likely preceded (and possibly triggered) by a ligand-induced conformational change, which could bring the catalytic dyad into the proximity to effect proton transfer.

Structural insights and binding of a natural ligand, succinic acid with serine and cysteine proteases

In the age of growing infectious diseases, there is a great demand for new inhibitors which can exhibit minimum side effects. Owing to the importance of proteases in life cycle and invasion, they have been projected as attractive targets for structure based drug designing against microbes including viruses. Here we report the inhibitory activity of a well known natural compound succinic acid against both serine and cysteine proteases. The ligand is found co-crystallized with Bovine pancreatic trypsin in one of our crystallization trials and the diffraction data up to1.9 Å reveal its interactions with the catalytic triad residues Histidine 57 and Serine 195. Binding of the ligand with these proteases have been validated using caseinolysis inhibition. With trypsin, ITC analysis showed tight binding of the ligand, resulting in change in Gibb's free energy (ΔG) by −20.31 kJ/mol. To understand the existence of succinic acid at the active site, molecular docking was performed and it revealed binding of it with trypsin and papain at corresponding active sites. This dual inhibitory activity of natural ligand, succinic acid can be accounted for the recent reports on anti-viral property of plant extracts where dicarboxilic fatty acids are normally abundant.

Metal Dependence of the Xylose Isomerase from Piromyces sp. E2 Explored by Activity Profiling and Protein Crystallography.

Xylose isomerase from Piromyces sp. E2 (PirXI) can be used to equip Saccharomyces cerevisiae with the capacity to ferment xylose to ethanol. The biochemical properties and structure of the enzyme have not been described even though its metal content, catalytic parameters, and expression level are critical for rapid xylose utilization. We have isolated the enzyme after high-level expression in Escherichia coli, analyzed the metal dependence of its catalytic properties, and determined 12 crystal structures in the presence of different metals, substrates, and substrate analogues. The activity assays revealed that various bivalent metals can activate PirXI for xylose isomerization. Among these metals, Mn2+ is the most favorable for catalytic activity. Furthermore, the enzyme shows the highest affinity for Mn2+, which was established by measuring the activation constants (Kact) for different metals. Metal analysis of the purified enzyme showed that in vivo the enzyme binds a mixture of metals that is determined by metal availability as well as affinity, indicating that the native metal composition can influence activity. The crystal structures show the presence of an active site similar to that of other xylose isomerases, with a d-xylose binding site containing two tryptophans and a catalytic histidine, as well as two metal binding sites that are formed by carboxylate groups of conserved aspartates and glutamates. The binding positions and conformations of the metal-coordinating residues varied slightly for different metals, which is hypothesized to contribute to the observed metal dependence of the isomerase activity.