David C. Cantu

Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

BackgroundAcyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids.ResultsTo explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family.ConclusionThese acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.

Thioesterases: A new perspective based on their primary and tertiary structures

Thioesterases (TEs) are classified into EC 3.1.2.1 through EC 3.1.2.27 based on their activities on different substrates, with many remaining unclassified (EC 3.1.2.–). Analysis of primary and tertiary structures of known TEs casts a new light on this enzyme group. We used strong primary sequence conservation based on experimentally proved proteins as the main criterion, followed by verification with tertiary structure superpositions, mechanisms, and catalytic residue positions, to accurately define TE families. At present, TEs fall into 23 families almost completely unrelated to each other by primary structure. It is assumed that all members of the same family have essentially the same tertiary structure; however, TEs in different families can have markedly different folds and mechanisms. Conversely, the latter sometimes have very similar tertiary structures and catalytic mechanisms despite being only slightly or not at all related by primary structure, indicating that they have common distant ancestors and can be grouped into clans. At present, four clans encompass 12 TE families. The new constantly updated ThYme (Thioester‐active enzYmes) database contains TE primary and tertiary structures, classified into families and clans that are different from those currently found in the literature or in other databases. We review all types of TEs, including those cleaving CoA, ACP, glutathione, and other protein molecules, and we discuss their structures, functions, and mechanisms.

ThYme: a database for thioester-active enzymes

The ThYme (Thioester-active enzYme; http://www.enzyme.cbirc.iastate.edu) database has been constructed to bring together amino acid sequences and 3D (tertiary) structures of all the enzymes constituting the fatty acid synthesis and polyketide synthesis cycles. These enzymes are active on thioester-containing substrates, specifically those that are parts of the acyl-CoA synthase, acyl-CoA carboxylase, acyl transferase, ketoacyl synthase, ketoacyl reductase, hydroxyacyl dehydratase, enoyl reductase and thioesterase enzyme groups. These groups have been classified into families, members of which are similar in sequences, tertiary structures and catalytic mechanisms, implying common protein ancestry. ThYme is continually updated as sequences and tertiary structures become available.

CO Oxidation on Au/TiO2: Condition-Dependent Active Sites and Mechanistic Pathways

We present results of ab initio electronic structure and molecular dynamics simulations (AIMD), as well as a microkinetic model of CO oxidation catalyzed by TiO2 supported Au nanocatalysts. A coverage-dependent microkinetic analysis, based on energetics obtained with density functional methods, shows that the dominant kinetic pathway, activated oxygen species, and catalytic active sites are all strongly depended on both temperature and oxygen partial pressure. Under oxidizing conditions and T < 400 K, the prevalent pathway involves a dynamic single atom catalytic mechanism. This reaction is catalyzed by a transient Au-CO species that migrates from the Au-cluster onto a surface oxygen adatom. It subsequently reacts with the TiO2 support via a Mars van Krevelen mechanism to form CO2 and finally the Au atom reintegrates back into the gold cluster to complete the catalytic cycle. At 300 ≤ T ≤ 600 K, oxygen-bound single Oad-Au(+)-CO sites and the perimeter Au-sites of the nanoparticle work in tandem to optimally catalyze the reaction. Above 600 K, a variety of alternate pathways associated with both single-atom and the perimeter sites of the Au nanoparticle are found to be active. Under low oxygen pressures, Oad-Au(+)-CO species can be a source of catalyst deactivation and the dominant pathway involves only Au-perimeter sites. A detailed comparison of the current model and the existing literature resolves many apparent inconsistencies in the mechanistic interpretations.

Structural classification and properties of ketoacyl synthases

Ketoacyl synthases (KSs) catalyze condensing reactions combining acyl‐CoA or acyl‐acyl carrier protein (acyl‐ACP) with malonyl‐CoA to form 3‐ketoacyl‐CoA or with malonyl‐ACP to form 3‐ketoacyl‐ACP. In each case, the resulting acyl chain is two carbon atoms longer than before, and CO2 and either CoA or ACP are formed. KSs also join other activated molecules in the polyketide synthesis cycle. Our classification of KSs by their primary and tertiary structures instead of by their substrates and the reactions that they catalyze enhances insights into this enzyme group. KSs fall into five families separated by their characteristic primary structures, each having members with the same catalytic residues, mechanisms, and tertiary structures. KS1 members, overwhelmingly named 3‐ketoacyl‐ACP synthase III or its variants, are produced predominantly by bacteria. Members of KS2 are mainly produced by plants, and they are usually long‐chain fatty acid elongases/condensing enzymes and 3‐ketoacyl‐CoA synthases. KS3, a very large family, is composed of bacterial and eukaryotic 3‐ketoacyl‐ACP synthases I and II, often found in multidomain fatty acid and polyketide synthases. Most of the chalcone synthases, stilbene synthases, and naringenin‐chalcone synthases in KS4 are from eukaryota. KS5 members are all from eukaryota, most are produced by animals, and they are mainly fatty acid elongases. All families except KS3 are split into subfamilies whose members have statistically significant differences in their primary structures. KS1 through KS4 appear to be part of the same clan. KS sequences, tertiary structures, and family classifications are available on the continuously updated ThYme (Thioester‐active enzYme) database.

Water-Lean Solvents for Post-Combustion CO2 Capture: Fundamentals, Uncertainties, Opportunities, and Outlook

This review is designed to foster the discussion regarding the viability of postcombustion CO2 capture by water-lean solvents, by separating fact from fiction for both skeptics and advocates. We highlight the unique physical and thermodynamic properties of notable water-lean solvents, with a discussion of how such properties could translate to efficiency gains compared to aqueous amines. The scope of this review ranges from the purely fundamental molecular-level processes that govern solvent behavior to bench-scale testing, through process engineering and projections of process performance and cost. Key discussions of higher than expected CO2 mass transfer, water tolerance, and compatibility with current infrastructure are presented along with current limitations and suggested areas where further solvent development is needed. We conclude with an outlook of the status of the field and assess the viability of water-lean solvents for postcombustion CO2 capture.

Dimerization Induced Deprotonation of Water on RuO2(110).

RuO2 has proven to be indispensable as a co-catalyst in numerous systems designed for photocatalytic water splitting. In this study, we have carried out a detailed mechanistic study of water behavior on the most stable RuO2 face, RuO2(110), by employing variable-temperature scanning tunneling microscopy and density functional theory calculations. We show that water monomers adsorb molecularly on Ru sites, become mobile above 238 K, diffuse along the Ru rows, and form water dimers. The onset for dimer diffusion is observed at ∼277 K, indicating a significantly higher diffusion barrier than that for monomers. More importantly, we find that water dimers deprotonate readily to form Ru-bound H3O2 and bridging OH species. The observed behavior is compared and contrasted with that observed for water on isostructural rutile TiO2(110).

Dynamic Acid/Base Equilibrium in Single Component Switchable Ionic Liquids and Consequences on Viscosity.

The deployment of transformational nonaqueous CO2-capture solvent systems is encumbered by high viscosities even at intermediate uptakes. Using single-molecule CO2 binding organic liquids as a prototypical example, we present key molecular features that control bulk viscosity. Fast CO2-uptake kinetics arise from close proximity of the alcohol and amine sites involved in CO2 binding in a concerted fashion, resulting in a Zwitterion containing both an alkyl-carbonate and a protonated amine. The population of internal hydrogen bonds between the two functional groups determines the solution viscosity. Unlike the ion pair interactions in ionic liquids, these observations are novel and specific to a hydrogen-bonding network that can be controlled by chemically tuning single molecule CO2 capture solvents. We present a molecular design strategy to reduce viscosity by shifting the proton transfer equilibrium toward a neutral acid/amine species, as opposed to the ubiquitously accepted zwitterionic state. The molecular design concepts proposed here are readily extensible to other CO2 capture technologies.

Molecular Mechanism of a Hotdog‐Fold Acyl‐CoA Thioesterase

Thioesterases are enzymes that hydrolyze thioester bonds between a carbonyl group and a sulfur atom. They catalyze key steps in fatty acid biosynthesis and metabolism, as well as polyketide biosynthesis. The reaction molecular mechanism of most hotdog-fold acyl-CoA thioesterases remains unknown, but several hypotheses have been put forward in structural and biochemical investigations. The reaction of a human thioesterase (hTHEM2), representing a thioesterase family with a hotdog fold where a coenzyme A moiety is cleaved, was simulated by quantum mechanics/molecular mechanics metadynamics techniques to elucidate atomic and electronic details of its mechanism, its transition-state conformation, and the free energy landscape of the process. A single-displacement acid-base-like mechanism, in which a nucleophilic water molecule is activated by an aspartate residue acting as a base, was found, confirming previous experimental proposals. The results provide unambiguous evidence of the formation of a tetrahedral-like transition state. They also explain the roles of other conserved active-site residues during the reaction, especially that of a nearby histidine/serine pair that protonates the thioester sulfur atom, the participation of which could not be elucidated from mutation analyses alone.

Structural Rearrangement of Au–Pd Nanoparticles under Reaction Conditions: An ab Initio Molecular Dynamics Study

The structure, composition, and atomic distribution of nanoalloys under operating conditions are of significant importance for their catalytic activity. In the present work, we use ab initio molecular dynamics simulations to understand the structural behavior of Au-Pd nanoalloys supported on rutile TiO2 under different conditions. We find that the Au-Pd structure is strongly dependent on the redox properties of the support, originating from strong metal-support interactions. Under reducing conditions, Pd atoms are inclined to move toward the metal/oxide interface, as indicated by a significant increase of Pd-Ti bonds. This could be attributed to the charge localization at the interface that leads to Coulomb attractions to positively charged Pd atoms. In contrast, under oxidizing conditions, Pd atoms would rather stay inside or on the exterior of the nanoparticle. Moreover, Pd atoms on the alloy surface can be stabilized by hydrogen adsorption, forming Pd-H bonds, which are stronger than Au-H bonds. Our work offers critical insights into the structure and redox properties of Au-Pd nanoalloy catalysts under working conditions.