Large Active Site Research Articles

What Determines the Selectivity of Arginine Dihydroxylation by the Nonheme Iron Enzyme OrfP?

The nonheme iron enzyme OrfP reacts with l-Arg selectively to form the 3R,4R-dihydroxyarginine product, which in mammals can inhibit the nitric oxide synthase enzymes involved in blood pressure control. To understand the mechanisms of dioxygen activation of l-Arg by OrfP and how it enables two sequential oxidation cycles on the same substrate, we performed a density functional theory study on a large active site cluster model. We show that substrate binding and positioning in the active site guides a highly selective reaction through C3 -H hydrogen atom abstraction. This happens despite the fact that the C3 -H and C4 -H bond strengths of l-Arg are very similar. Electronic differences in the two hydrogen atom abstraction pathways drive the reaction with an initial C3 -H activation to a low-energy 5 σ-pathway, while substrate positioning destabilizes the C4 -H abstraction and sends it over the higher-lying 5 π-pathway. We show that substrate and monohydroxylated products are strongly bound in the substrate binding pocket and hence product release is difficult and consequently its lifetime will be long enough to trigger a second oxygenation cycle.

Lignin Biodegradation by a Cytochrome P450 Enzyme: A Computational Study into Syringol Activation by GcoA.

A recently characterized cytochrome P450 isozyme GcoA activates lignin components through a selective O‐demethylation or alternatively an acetal formation reaction. These are important reactions in biotechnology and, because lignin is readily available; it being the main component in plant cell walls. In this work we present a density functional theory study on a large active site model of GcoA to investigate syringol activation by an iron(IV)‐oxo heme cation radical oxidant (Compound I) leading to hemiacetal and acetal products. Several substrate‐binding positions were tested and full energy landscapes calculated. The study shows that substrate positioning determines the product distributions. Thus, with the phenol group pointing away from the heme, an O‐demethylation is predicted, whereas an initial hydrogen‐atom abstraction of the weak phenolic O‐H group would trigger a pathway leading to ring‐closure to form acetal products. Predictions on how to engineer P450 GcoA to get more selective product distributions are given.

Catalytic Mechanism of Aromatic Nitration by Cytochrome P450 TxtE: Involvement of a Ferric-Peroxynitrite Intermediate

The cytochromes P450 are heme-dependent enzymes that catalyze many vital reaction processes in the human body related to biodegradation and biosynthesis. They typically act as mono-oxygenases; however, the recently discovered P450 subfamily TxtE utilizes O2 and NO to nitrate aromatic substrates such as L-tryptophan. A direct and selective aromatic nitration reaction may be useful in biotechnology for the synthesis of drugs or small molecules. Details of the catalytic mechanism are unknown, and it has been suggested that the reaction should proceed through either an iron(III)-superoxo or an iron(II)-nitrosyl intermediate. To resolve this controversy, we used stopped-flow kinetics to provide evidence for a catalytic cycle where dioxygen binds prior to NO to generate an active iron(III)-peroxynitrite species that is able to nitrate l-Trp efficiently. We show that the rate of binding of O2 is faster than that of NO and also leads to l-Trp nitration, while little evidence of product formation is observed from the iron(II)-nitrosyl complex. To support the experimental studies, we performed density functional theory studies on large active site cluster models. The studies suggest a mechanism involving an iron(III)-peroxynitrite that splits homolytically to form an iron(IV)-oxo heme (Compound II) and a free NO2 radical via a small free energy of activation. The latter activates the substrate on the aromatic ring, while compound II picks up the ipso-hydrogen to form the product. The calculations give small reaction barriers for most steps in the catalytic cycle and, therefore, predict fast product formation from the iron(III)-peroxynitrite complex. These findings provide the first detailed insight into the mechanism of nitration by a member of the TxtE subfamily and highlight how the enzyme facilitates this novel reaction chemistry.

Synthesis and electrorheological properties of hierarchical and core-shell MoS2@TiO2 nanocomposite

MoS2@TiO2 nanocomposite with core-shell and hierarchical structure was synthesized using a simple and effective two-step method. First, 3D radically oriented MoS2 nanospheres were initially synthesized using a simple and convenient hydrothermal method to provide a large surface and active site for the uniform TiO2 growth on MoS2. Then, the structure, morphology, composition and electrorheological (ER) properties were characterised using an X-ray powder diffractometer, Fourier infrared spectrometer, transmission electron microscopy, scanning electron microscope with an attached energy-dispersive spectrometer, X-ray photoelectron spectroscopy and ER rheometer. The MoS2@TiO2 nanocomposite was dispersed into insulating silicone oil to form an ER fluid at a concentration of 10 ​wt %, which shown an enhanced ER efficiency by combining the useful of two dimensional MoS2 multistage structures and coating TiO2 shell to control conductivity. The large dielectric constant mismatch and short relaxation time of ER fluid was observed, which resulted in the creation of multiple polarizations and enhanced ER properties.

It takes two to tango - The case of thebaine 6-O-demethylase

Thebaine 6-O-demethylase (T6ODM) is an Fe(II)/2-oxoglutarate-dependent dioxygenase catalysing two oxidative O-demethylation reactions in morphine biosynthesis. Its crystal structure revealed a large active site pocket which is at least two times larger than necessary to accommodate a substrate (thebaine or oripavine) molecule. Since so far no crystal structures have been obtained for enzyme-substrate complex, which is necessary to explain the enzyme regiospecificity towards the C6-bound methoxy group, in this work we used computational methods and multi-parametric surface plasmon resonance measurements to elucidate the most likely structure of this complex and the reaction mechanism starting therefrom. Results of simulations and experiments unanimously indicate that the enzyme-substrate complex of T6ODM has a 1:2 stoichiometry. The key residues responsible for substrate binding are: Val-128, Glu-133, Met-150 and Agr-219 for the substrate in the distal position, and Asp-144, Leu-235 and Leu-353 for the proximal substrate molecule. QM/MM and DFT calculations show that the oxo ligand is bound trans to His-295 and the enzyme catalyzes hydroxylation of the C6-bound methoxy group according to the established rebound mechanism. The final stage of the demethylation reaction, which includes deformylation and enol-keton tautomerization steps, is most likely catalysed by water molecules and takes place in the solvent.

Larger active site in an ancestral hydroxynitrile lyase increases catalytically promiscuous esterase activity.

Hydroxynitrile lyases (HNL's) belonging to the α/β-hydrolase-fold superfamily evolved from esterases approximately 100 million years ago. Reconstruction of an ancestral hydroxynitrile lyase in the α/β-hydrolase fold superfamily yielded a catalytically active hydroxynitrile lyase, HNL1. Several properties of HNL1 differ from the modern HNL from rubber tree (HbHNL). HNL1 favors larger substrates as compared to HbHNL, is two-fold more catalytically promiscuous for ester hydrolysis (p-nitrophenyl acetate) as compared to mandelonitrile cleavage, and resists irreversible heat inactivation to 35 °C higher than for HbHNL. We hypothesized that the x-ray crystal structure of HNL1 may reveal the molecular basis for the differences in these properties. The x-ray crystal structure solved to 1.96-Å resolution shows the expected α/β-hydrolase fold, but a 60% larger active site as compared to HbHNL. This larger active site echoes its evolution from esterases since related esterase SABP2 from tobacco also has a 38% larger active site than HbHNL. The larger active site in HNL1 likely accounts for its ability to accept larger hydroxynitrile substrates. Site-directed mutagenesis of HbHNL to expand the active site increased its promiscuous esterase activity 50-fold, consistent with the larger active site in HNL1 being the primary cause of its promiscuous esterase activity. Urea-induced unfolding of HNL1 indicates that it unfolds less completely than HbHNL (m-value = 0.63 for HNL1 vs 0.93 kcal/mol·M for HbHNL), which may account for the ability of HNL1 to better resist irreversible inactivation upon heating. The structure of HNL1 shows changes in hydrogen bond networks that may stabilize regions of the folded structure.

Functionalized CoFe2O4/lamellar mesopore silica anchored to melamine nanocomposite as a novel catalyst for synthesis of 4H‐chromenes under mild conditions

In this research, it was displayed an efficient method for the one‐pot reaction of cyclohexanone, benzaldehyde and malononitrile for the synthesis of 4H‐chromenes by using CoFe2O4/lamellar mesopore silica anchored to melamine as a magnetic nanocatalyst. This nanocatalyst was prepared in several steps and discriminated by XRD, FT‐IR, SEM, VSM, TGA and BET techniques. The catalyst has a large active base site that has functionalized in both the surface and the pore of nanostructure. The advantages of magnetic nanocatalyst were simple accessible, heterogeneous nanocatalyst, easy work up and reusability. The various derivatives of 4H‐chromenes were synthesized in the presence of CoFe2O4/lamellar mesopore silica/melamine magnetic nanocatalyst with the excellent yields and appropriate times. The products were identified by the melting point, FT‐IR,1H NMR,13C NMR and C.H.N techniques.

Replacement of Ca by Ni in a Perovskite Titanate to Yield a Novel Perovskite Exsolution Architecture for Oxygen‐Evolution Reactions

AbstractFor efficient catalysis and electrocatalysis well‐designed, high‐surface‐area support architectures covered with highly dispersed metal nanoparticles with good catalyst‐support interactions are required. In situ grown Ni nanoparticles on perovskites have been recently reported to enhance catalytic activities in high‐temperature systems such as solid oxide cells (SOCs). However, the micrometer‐scale primary particles prepared by conventional solid‐state reactions have limited surface area and tend to retain much of the active catalytic element within the bulk, limiting efficacy of such exsolution processes in low‐temperature systems. Here, a new, highly efficient, solvothermal route is demonstrated to exsolution from smaller scale primary particles. Furthermore, unlike previous reports of B‐site exsolution, it seems that the metal nanoparticles are exsolved from the A‐site of these perovskites. The catalysts show large active site areas and strong metal‐support interaction (SMSI), leading to ≈26% higher geometric activity (25 times higher mass activity with 1.4 V of Eon‐set) and stability for oxygen‐evolution reaction (OER) with only 0.72 µg base metal contents compared to typical 20 wt% Ni/C and even commercial 20 wt% Ir/C. The findings obtained here demonstrate the potential design and development of heterogeneous catalysts in various low‐temperature electrochemical systems including alkaline fuel cells and metal–air batteries.

How external perturbations affect the chemoselectivity of substrate activation by cytochrome P450 OleTJE.

Cytochrome P450 enzymes are versatile biocatalysts found in most forms of life. Generally, the cytochrome P450s react with dioxygen and hence are haem-based mono-oxygenases; however, in specific isozymes, H2O2 rather than O2 is used and these P450s act as peroxygenases. The P450 OleTJE is a peroxygenase that binds long to medium chain fatty acids and converts them to a range of products originating from Cα-hydroxylation, Cβ-hydroxylation, Cα-Cβ desaturation and decarboxylation of the substrate. There is still controversy regarding the details of the reaction mechanism of P450 OleTJE; how the products are formed and whether the product distributions can be influenced by external perturbations. To gain further insights into the structure and reactivity of P450 OleTJE, we set up a range of large active site model complexes as well as full enzymatic structures and did a combination of density functional theory studies and quantum mechanics/molecular mechanics calculations. In particular, the work focused on the mechanisms leading to these products under various reaction conditions. Thus, for a small cluster model, we find a highly selective Cα-hydroxylation pathway that is preferred over Cβ-H hydrogen atom abstraction by at least 10 kcal mol-1. Introduction of polar residues to the model, such as an active site protonated histidine residue or through external electric field effects, lowers the Cβ-H hydrogen atom abstraction barriers are lowered, while a full QM/MM model brings the Cα-H and Cβ-H hydrogen atom abstraction barriers within 1 kcal mol-1. Our studies; therefore, implicate that environmental effects in the second-coordination sphere can direct and guide selectivities in enzymatic reaction mechanisms.

Targeting a Large Active Site: Structure-Based Design of Nanomolar Inhibitors of Trypanosoma brucei Trypanothione Reductase.

Trypanothione reductase (TR) plays a key role in the unique redox metabolism of trypanosomatids, the causative agents of human African trypanosomiasis (HAT), Chagas' disease, and leishmaniases. Introduction of a new, lean propargylic vector to a known class of TR inhibitors resulted in the strongest reported competitive inhibitor of Trypanosoma (T.) brucei TR, with an inhibition constant Ki of 73 nm, which is fully selective against human glutathione reductase (hGR). The best ligands exhibited in vitro IC50 values (half-maximal inhibitory concentration) against the HAT pathogen, T. brucei rhodesiense, in the mid-nanomolar range, reaching down to 50 nm. X-Ray co-crystal structures confirmed the binding mode of the ligands and revealed the presence of a HEPES buffer molecule in the large active site. Extension of the propargylic vector, guided by structure-based design, to replace the HEPES buffer molecule should give inhibitors with low nanomolar Ki and IC50 values for in vivo studies.

Synthesis of ultrathin Co2AlO4 nanosheets with oxygen vacancies for enhanced electrocatalytic oxygen evolution

In this work, we reported the synthesis of two-dimensional spinel structure of ultrathin Co2AlO4 nanosheets via dealloying and subsequent annealing processes. Oxygen vacancy defects were further introduced into Co2AlO4 nanosheets by a mild solvothermal reduction method, resulting in large electrochemical surface area and high active site densities, making the related Co atoms get electrons, and producing more empty orbitals. The positive charge of Co and Al atoms adjacent to the O vacancies in VO-rich Co2AlO4 reduced significantly, that is, more electrons are concentrated on the Co and Al atoms. Those electrons closed to the Fermi level have a promoting effect during the H2O activation. As a result, the obtained ultrathin Co2AlO4 nanosheets with oxygen vacancies show a low overpotential of 280 mV at the current density of 10 mA cm−2 and a small Tafel slope of 70.98 mV dec−1. Moreover, it also displays a remarkable stability in alkaline solution, which is superior to most of the reported Co3O4 electrocatalysts. The present work paves a new way to achieve efficient new energetic materials for sustainable community.

Flavonol biosynthesis by nonheme iron dioxygenases: A computational study into the structure and mechanism

Plants produce flavonol compounds for vital functions regarding plant growth, fruit and flower colouring as well as fruit ripening processes. Several of these biosynthesis steps are stereo- and regioselective and are being carried out by nonheme iron enzymes. Using density functional theory calculations on a large active site model complex of flavanone-3β-hydroxylase (FHT), we established the mechanism for conversion of naringenin to its dihydroflavonol, which is a key step in the mechanism of flavonol biosynthesis. The reaction starts with dioxygen binding to the iron(II) centre and a reaction with α-ketoglutarate co-substrate gives succinate, an iron(IV)-oxo species and CO2 with large exothermicity and small reaction barriers. The rate-determining reaction step in the mechanism; however, is hydrogen atom abstraction of an aliphatic CH bond by the iron(IV)-oxo species. We identify a large kinetic isotope effect for the replacement of the transferring hydrogen atom by deuterium. In a final step the OH and substrate radicals combine to form the alcohol product with a barrier of several kcal mol−1. We show that the latter is the result of geometric constraints in the active site pocket. Furthermore, the calculations show that a weak tertiary CH bond is shielded from the iron(IV)-oxo species in the substrate binding position and therefore the enzyme is able to activate a stronger CH bond. As such, the flavanone-3β-hydroxylase enzyme reacts regioselectively with one specific CH bond of naringenin by avoiding activation of weaker bonds through tight substrate and oxidant positioning.

Active-site differences between substrate-free and ritonavir-bound cytochrome P450 (CYP) 3A5 reveal plasticity differences between CYP3A5 and CYP3A4

Cytochrome P450 (CYP) 3A4 is a major contributor to hepatic drug and xenobiotic metabolism in human adults. The related enzyme CYP3A5 is also expressed in adult liver and has broader age and tissue distributions. However, CYP3A5 expression is low in most Caucasians because of the prevalence of an allele that leads to an incorrectly spliced mRNA and premature termination of translation. When expressed, CYP3A5 expands metabolic capabilities and can augment CYP3A4-mediated drug metabolism, thereby reducing drug efficacy and potentially requiring dose adjustments. The extensive role of CYP3A4 in drug metabolism reflects in part the plasticity of the substrate-free enzyme to enlarge its active site and accommodate very large substrates. We have previously shown that the structure of the CYP3A5-ritonavir complex differs substantially from that of the CYP3A4-ritonavir complex. To better understand whether these differences are conserved in other CYP3A5 structures and how they relate to differential plasticity, we determined the X-ray crystallographic structure of the CYP3A5 substrate-free complex to 2.20 Å resolution. We observed that this structure exhibits a much larger active site than substrate-free CYP3A4 and displays an open substrate access channel. This reflected in part a lower trajectory of the helix F-F' connector in CYP3A4 and more extensive π-CH interactions between phenylalanine residues forming the roof of the active-site cavity than in CYP3A5. Comparison with the CYP3A5-ritonavir complex confirmed conserved CYP3A5 structural features and indicated differences in plasticity between CYP3A4 and CYP3A5 that favor alternative ritonavir conformations.

DNA Aptamers Targeting BACE1 Reduce Amyloid Levels and Rescue Neuronal Deficiency in Cultured Cells

β-amyloid (Aβ) plays an essential role in the pathogenesis of Alzheimer’s disease (AD). Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is indispensable for Aβ production, and knockout of BACE1 has no overt phenotypes in mouse. Thus, fine modulation of BACE1 may be a safe and effective treatment for AD patients. However, the large active site of BACE1 makes it challenging to target BACE1 with classical small-molecule inhibitors. DNA aptamer can have high affinity and specificity against diverse targets, and it provides an alternative strategy to target BACE1. In this study, we used a novel cell-systematic evolution of ligands by exponential enrichment (SELEX) strategy to select specific DNA aptamers optimized to target BACE1 under physiological status. After 17 rounds of selection, we identified two DNA aptamers against BACE1: BI1 and BI2. The identified aptamers interacted with BACE1 in pull-down assay, inhibited BACE1 activity in in vitro fluorescence resonance energy transfer (FRET) assay and HEK293-APP stable cell line, reduced Aβ in the culture medium of HEK293-amyloid protein precursor (APP) stable cell line and APP-PS1 primary cultured neurons, and rescued Aβ-induced neuronal deficiency in APP-PS1 primary cultured neurons. In contrast, the identified aptamers had no effect on α- or γ-secretase. In addition, cholesteryl tetraetylene glycol (TEG) modification further improved the potency of the identified aptamers. Our study suggests that it is feasible and effective to target BACE1 with DNA aptamers, and the therapeutic potential of the identified aptamers deserves further investigation.

Electrocatalytic CO₂ Reduction: From Homogeneous Catalysts to Heterogeneous-Based Reticular Chemistry.

CO2, emitted mainly from fossil fuel combustion, is one of the major greenhouse gases. CO2 could be converted into more valuable chemical feedstocks including CO, HCOOH, HCHO, CH3OH, or CH4. To reduce CO2, catalysts were designed and their unique characteristics were utilized based on types of reaction processes, including catalytic hydrogenation, complex metal hydrides, photocatalysis, biological reduction, and electrochemical reduction. Indeed, the electroreduction method has received much consideration lately due to the simple operation, as well as environmentally friendly procedures that need to be optimized by both of the catalysts and the electrochemical process. In the past few decades, we have witnessed an explosion in development in materials science—especially in regards to the porous crystalline materials based on the strong covalent bond of the organic linkers containing light elements (Covalent organic frameworks, COFs), as well as the hybrid materials that possess organic backbones and inorganic metal-oxo clusters (Metal-organic frameworks, MOFs). Owing to the large surface area and high active site density that belong to these tailorable structures, MOFs and COFs can be applied to many practical applications, such as gas storage and separation, drug release, sensing, and catalysis. Beyond those applications, which have been abundantly studied since the 1990s, CO2 reduction catalyzed by reticular and extended structures of MOFs or COFs has been more recently turned to the next step of state-of-the-art application. In this perspective, we highlight the achievement of homogeneous catalysts used for CO2 electrochemical conversion and contrast it with the advances in new porous catalyst-based reticular chemistry. We then discuss the role of new catalytic systems designed in light of reticular chemistry in the heterogeneous-catalyzed reduction of CO2.

Structure and role for active site lid of lactate monooxygenase from Mycobacterium smegmatis.

Lactate monooxygenase (LMO) catalyzes the FMN-dependent "coupled" oxidation of lactate and O2 to acetate, carbon dioxide, and water, involving pyruvate and hydrogen peroxide as enzyme-bound intermediates. Other α-hydroxy acid oxidase family members follow an "uncoupled pathway," wherein the α-keto acid product quickly dissociates before the reduced flavin reacts with oxygen. Here, we report the structures of Mycobacterium smegmatis wild-type LMO and a wild-type-like C203A variant at 2.1 Å and 1.7 Å resolution, respectively. The overall LMO fold and active site organization, including a bound sulfate mimicking substrate, resemble those of other α-hydroxy acid oxidases. Based on structural similarity, LMO is similarly distant from lactate oxidase, glycolate oxidase, mandelate dehydrogenase, and flavocytochrome b2 and is the first representative enzyme of its type. Comparisons with other α-hydroxy acid oxidases reveal that LMO has a longer and more compact folded active site loop (Loop 4), which is known in related flavoenzymes to undergo order/disorder transitions to allow substrate/product binding and release. We propose that LMO's Loop 4 has an enhanced stability that is responsible for the slow product release requisite for the coupled pathway. We also note electrostatic features of the LMO active site that promote substrate binding. Whereas the physiological role of LMO remains unknown, we document what can currently be assessed of LMO's distribution in nature, including its unexpected occurrence, presumably through horizontal gene transfer, in halophilic archaea and in a limited group of fungi of the genus Beauveria. BROAD STATEMENT OF IMPACT: This first crystal structure of the FMN-dependent α-hydroxy acid oxidase family member lactate monooxygenase (LMO) reveals it has a uniquely large active site lid that we hypothesize is stable enough to explain the slow dissociation of pyruvate that leads to its "coupled" oxidation of lactate and O2 to produce acetate, carbon dioxide, and water. Also, the relatively widespread distribution of putative LMOs supports their importance and provides new motivation for their further study.

Toward Understanding the Chemistry and Biology of 1-Deoxy-d-xylulose 5-Phosphate (DXP) Synthase: A Unique Antimicrobial Target at the Heart of Bacterial Metabolism.

Antibiotics are the cornerstone of modern healthcare. The 20th century discovery of sulfonamides and β-lactam antibiotics altered human society immensely. Simple bacterial infections were no longer a leading cause of morbidity and mortality, and antibiotic prophylaxis greatly reduced the risk of infection from surgery. The current healthcare system requires effective antibiotics to function. However, antibiotic-resistant infections are becoming increasingly prevalent, threatening the emergence of a postantibiotic era. To prevent this public health crisis, antibiotics with novel modes of action are needed. Currently available antibiotics target just a few cellular processes to exert their activity: DNA, RNA, protein, and cell wall biosynthesis. Bacterial central metabolism is underexploited, offering a wealth of potential new targets that can be pursued toward expanding the armamentarium against microbial infections. Discovered in 1997 as the first enzyme in the methylerythritol phosphate (MEP) pathway, 1-deoxy-d-xylulose 5-phosphate (DXP) synthase is a thiamine diphosphate (ThDP)-dependent enzyme that catalyzes the decarboxylative condensation of pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) to form DXP. This five-carbon metabolite feeds into three separate essential pathways for bacterial central metabolism: ThDP synthesis, pyridoxal phosphate (PLP) synthesis, and the MEP pathway for isoprenoid synthesis. While it has long been identified as a target for the development of antimicrobial agents, limited progress has been made toward developing selective inhibitors of the enzyme. This Account highlights advances from our lab over the past decade to understand this important and unique enzyme. Unlike all other known ThDP-dependent enzymes, DXP synthase uses a random-sequential mechanism that requires the formation of a ternary complex prior to decarboxylation of the lactyl-ThDP intermediate. Its large active site accommodates a variety of acceptor substrates, lending itself to a number of alternative activities, such as the production of α-hydroxy ketones, hydroxamates, amides, acetolactate, and peracetate. Knowledge gained from mechanistic and substrate-specificity studies has guided the development of selective inhibitors with antibacterial activity and provides a biochemical foundation toward understanding DXP synthase function in bacterial cells. Although it is a promising drug target, the centrality of DXP synthase in bacterial metabolism imparts specific challenges to assessing antibacterial activity of DXP synthase inhibitors, and the susceptibility of most bacteria to current DXP synthase inhibitors is remarkably culture-medium-dependent. Despite these challenges, the study of DXP synthase is poised to reveal the role of DXP synthase in bacterial metabolic adaptability during infection, ultimately providing a more complete picture of how inhibiting this crucial enzyme can be used to develop novel antibiotics.

The Critical E4 State of Nitrogenase Catalysis.

The reaction catalyzed by the nitrogenase enzyme involves breaking the stable triple bond of the dinitrogen molecule and is consequently considered among the most challenging reactions in biology. While many aspects regarding its atomic mechanism remain to be elucidated, a kinetic scheme established by David Lowe and Roger Thorneley has remained a gold standard for functional studies of the enzyme for more than 30 years. Recent three-dimensional structures of ligand-bound states of molybdenum- and vanadium-dependent nitrogenases have revealed the actual site of substrate binding on the large active site cofactors of this class of enzymes. The binding mode of an inhibitor and a reaction intermediate further substantiate a hypothesis by Seefeldt, Hoffman, and Dean that the activation of N2 is made possible by a reductive elimination of H2 that leaves the cofactor in a super-reduced state that can bind and reduce the inert N2 molecule. Here we discuss the immediate implications of the structurally observed mode of binding of small molecules to the enzyme with respect to the early stages of the Thorneley-Lowe mechanism of nitrogenase. Four consecutive single-electron reductions give rise to two bridging hydrides at the cluster surface that can recombine to eliminate H2 and enable the reduced cluster to bind its substrate in a bridging mode.

Biological Evaluation and X-ray Co-crystal Structures of Cyclohexylpyrrolidine Ligands for Trypanothione Reductase, an Enzyme from the Redox Metabolism of Trypanosoma.

The tropical diseases human African trypanosomiasis, Chagas disease, and the various forms of leishmaniasis are caused by parasites of the family of trypanosomatids. These protozoa possess a unique redox metabolism based on trypanothione and trypanothione reductase (TR), making TR a promising drug target. We report the optimization of properties and potency of cyclohexylpyrrolidine inhibitors of TR by structure-based design. The best inhibitors were freely soluble and showed competitive inhibition constants (Ki ) against Trypanosoma (T.) brucei TR and T. cruzi TR and in vitro activities (half-maximal inhibitory concentration, IC50 ) against these parasites in the low micromolar range, with high selectivity against human glutathione reductase. X-ray co-crystal structures confirmed the binding of the ligands to the hydrophobic wall of the "mepacrine binding site" with the new, solubility-providing vectors oriented toward the surface of the large active site.

A Haloalkane Dehalogenase from a Marine Microbial Consortium Possessing Exceptionally Broad Substrate Specificity.

The haloalkane dehalogenase enzyme DmmA was identified by marine metagenomic screening. Determination of its crystal structure revealed an unusually large active site compared to those of previously characterized haloalkane dehalogenases. Here we present a biochemical characterization of this interesting enzyme with emphasis on its structure-function relationships. DmmA exhibited an exceptionally broad substrate specificity and degraded several halogenated environmental pollutants that are resistant to other members of this enzyme family. In addition to having this unique substrate specificity, the enzyme was highly tolerant to organic cosolvents such as dimethyl sulfoxide, methanol, and acetone. Its broad substrate specificity, high overexpression yield (200 mg of protein per liter of cultivation medium; 50% of total protein), good tolerance to organic cosolvents, and a broad pH range make DmmA an attractive biocatalyst for various biotechnological applications.IMPORTANCE We present a thorough biochemical characterization of the haloalkane dehalogenase DmmA from a marine metagenome. This enzyme with an unusually large active site shows remarkably broad substrate specificity, high overexpression, significant tolerance to organic cosolvents, and activity under a broad range of pH conditions. DmmA is an attractive catalyst for sustainable biotechnology applications, e.g., biocatalysis, biosensing, and biodegradation of halogenated pollutants. We also report its ability to convert multiple halogenated compounds to corresponding polyalcohols.