Coevolutionary Information Captures Catalytic Functions and Reveals Divergent Roles of Terpene Synthase Interdomain Connections.

Inferring the historical and biophysical causes of diversity within protein families is a complex puzzle. A key to unraveling this problem is characterizing the rugged topography of sequence-function adaptive landscapes. Using biochemical data from a 2 = 512 combinatorial library of tobacco 5--aristolochene synthase (TEAS) mutants engineered to make the native major product of Egyptian henbane premnaspirodiene synthase (HPS) and a complementary 512 mutant HPS library, we address the question of how product specificity is controlled.

Visualizing the Interface of Biotin and Fatty Acid Biosynthesis through SuFEx Probes.

Site-specific covalent conjugation offers a powerful tool to identify and understand protein-protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink the acyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5'-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners.

Masked cerulenin enables a dual-site selective protein crosslink.

Protein-reactive natural products such as the fungal metabolite cerulenin are recognized for their value as therapeutic candidates, due to their ability to selectively react with catalytic residues within a protein active site or a complex of protein domains. Here, we explore the development of fatty-acid and polyketide-synthase probes by synthetically modulating cerulenin's functional moieties. Using a mechanism-based approach, we reveal unique reactivity within cerulenin and adapt it for fluorescent labeling and crosslinking of fatty-acid and iterative type-I polyketide synthases.

Mechanism-based cross-linking probes capture the Escherichia coli ketosynthase FabB in conformationally distinct catalytic states.

Ketosynthases (KSs) catalyse essential carbon-carbon bond-forming reactions in fatty-acid biosynthesis using a two-step, ping-pong reaction mechanism. In Escherichia coli, there are two homodimeric elongating KSs, FabB and FabF, which possess overlapping substrate selectivity. However, FabB is essential for the biosynthesis of the unsaturated fatty acids (UFAs) required for cell survival in the absence of exogenous UFAs.

Structural Basis of Stereospecific Vanadium-Dependent Haloperoxidase Family Enzymes in Napyradiomycin Biosynthesis.

Vanadium-dependent haloperoxidases (VHPOs) from bacteria differ from their counterparts in fungi, macroalgae, and other bacteria by catalyzing organohalogenating reactions with strict regiochemical and stereochemical control. While this group of enzymes collectively uses hydrogen peroxide to oxidize halides for incorporation into electron-rich organic molecules, the mechanism for the controlled transfer of highly reactive chloronium ions in the biosynthesis of napyradiomycin and merochlorin antibiotics sets the vanadium-dependent chloroperoxidases apart. Here we report high-resolution crystal structures of two homologous VHPO family members associated with napyradiomycin biosynthesis, NapH1 and NapH3, that catalyze distinctive chemical reactions in the construction of meroterpenoid natural products.

Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon.

Single-particle analysis (SPA) by cryo-electron microscopy (cryo-EM) is now a mainstream technique for high-resolution structural biology. Structure determination by SPA relies upon obtaining multiple distinct views of a macromolecular object vitrified within a thin layer of ice. Ideally, a collection of uniformly distributed random projection orientations would amount to all possible views of the object, giving rise to reconstructions characterized by isotropic directional resolution.

Plant-based CO drawdown and storage as SiC.

Since the 1950's the Earth's natural carbon cycle has not sufficiently sequestrated excess atmospheric CO contributed by human activities. CO levels rose above 400 ppm in 2013 and are forecasted to exceed 500 ppm by 2070, a level last experienced during the Paleogene period 25-65 MYA. While humanity benefits from the extraction and combustion of carbon from Earth's crust, we have overlooked the impact on global climate change.

Structure and mechanistic analyses of the gating mechanism of elongating ketosynthases.

Ketosynthases (KSs) catalyze carbon-carbon bond forming reactions in fatty acid synthases (FASs) and polyketide synthases (PKSs). KSs utilize a two-step ping pong kinetic mechanism to carry out an overall decarboxylative thio-Claisen condensation that can be separated into the transacylation and condensation reactions. In both steps, an acyl carrier protein (ACP) delivers thioester tethered substrates to the active sites of KSs.

Interfacial plasticity facilitates high reaction rate of FAS malonyl-CoA:ACP transacylase, FabD.

Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP-AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways.

Activity Mapping the Acyl Carrier Protein: Elongating Ketosynthase Interaction in Fatty Acid Biosynthesis.

Elongating ketosynthases (KSs) catalyze carbon-carbon bond-forming reactions during the committed step for each round of chain extension in both fatty acid synthases (FASs) and polyketide synthases (PKSs). A small α-helical acyl carrier protein (ACP) shuttles fatty acyl intermediates between enzyme active sites. To accomplish this task, the ACP relies on a series of dynamic interactions with multiple partner enzymes of FAS and associated FAS-dependent pathways.

Algal neurotoxin biosynthesis repurposes the terpene cyclase structural fold into an -prenyltransferase.

Prenylation is a common biological reaction in all domains of life wherein prenyl diphosphate donors transfer prenyl groups onto small molecules as well as large proteins. The enzymes that catalyze these reactions are structurally distinct from ubiquitous terpene cyclases that, instead, assemble terpenes via intramolecular rearrangements of a single substrate. Herein, we report the structure and molecular details of a new family of prenyltransferases from marine algae that repurposes the terpene cyclase structural fold for the -prenylation of glutamic acid during the biosynthesis of the potent neurochemicals domoic acid and kainic acid.

Modulation of auxin formation by the cytosolic phenylalanine biosynthetic pathway.

In plants, phenylalanine biosynthesis occurs via two compartmentally separated pathways. Overexpression of petunia chorismate mutase 2 (PhCM2), which catalyzes the committed step of the cytosolic pathway, increased flux in cytosolic phenylalanine biosynthesis, but paradoxically decreased the overall levels of phenylalanine and phenylalanine-derived volatiles. Concomitantly, the levels of auxins, including indole-3-acetic acid and its precursor indole-3-pyruvic acid, were elevated.

Gating mechanism of elongating β-ketoacyl-ACP synthases.

Carbon-carbon bond forming reactions are essential transformations in natural product biosynthesis. During de novo fatty acid and polyketide biosynthesis, β-ketoacyl-acyl carrier protein (ACP) synthases (KS), catalyze this process via a decarboxylative Claisen-like condensation reaction. KSs must recognize multiple chemically distinct ACPs and choreograph a ping-pong mechanism, often in an iterative fashion.

Dissecting modular synthases through inhibition: A complementary chemical and genetic approach.

Modular synthases, such as fatty acid, polyketide, and non-ribosomal peptide synthases (NRPSs), are sophisticated machineries essential in both primary and secondary metabolism. Various techniques have been developed to understand their genetic background and enzymatic abilities. However, uncovering the actual biosynthetic pathways remains challenging.

Substrate Specificity and Engineering of Mevalonate 5-Phosphate Decarboxylase.

A bifurcation of the mevalonate (MVA) pathway was recently discovered in bacteria of the Chloroflexi phylum. In this alternative route for the biosynthesis of isopentenylpyrophosphate (IPP), the penultimate step is the decarboxylation of ()-mevalonate 5-phosphate (()-MVAP) to isopentenyl phosphate (IP), which is followed by the ATP-dependent phosphorylation of IP to IPP catalyzed by isopentenyl phosphate kinase (IPK). Notably, the decarboxylation reaction is catalyzed by mevalonate 5-phosphate decarboxylase (MPD), which shares considerable sequence similarity with mevalonate diphosphate decarboxylase (MDD) of the classical MVA pathway.

Strigolactone perception and deactivation by a hydrolase receptor DWARF14.

The perception mechanism for the strigolactone (SL) class of plant hormones has been a subject of debate because their receptor, DWARF14 (D14), is an α/β-hydrolase that can cleave SLs. Here we show via time-course analyses of SL binding and hydrolysis by Arabidopsis thaliana D14, that the level of uncleaved SL strongly correlates with the induction of the active signaling state. In addition, we show that an AtD14 catalytic mutant that lacks enzymatic activity is still able to complement the atd14 mutant phenotype in an SL-dependent manner.

Contribution of isopentenyl phosphate to plant terpenoid metabolism.

Plant genomes encode isopentenyl phosphate kinases (IPKs) that reactivate isopentenyl phosphate (IP) via ATP-dependent phosphorylation, forming the primary metabolite isopentenyl diphosphate (IPP) used generally for isoprenoid/terpenoid biosynthesis. Therefore, the existence of IPKs in plants raises unanswered questions concerning the origin and regulatory roles of IP in plant terpenoid metabolism. Here, we provide genetic and biochemical evidence showing that IP forms during specific dephosphorylation of IPP catalysed by a subset of Nudix superfamily hydrolases.

Publisher Correction: Evolution of chalcone isomerase from a noncatalytic ancestor.

In the version of this article originally published, the number for the equal contributions footnote was missing for Miriam Kaltenbach and Jason R. Burke in the author list. The error has been corrected in the PDF and print versions of this article.

Evolution of chalcone isomerase from a noncatalytic ancestor.

The emergence of catalysis in a noncatalytic protein scaffold is a rare, unexplored event. Chalcone isomerase (CHI), a key enzyme in plant flavonoid biosynthesis, is presumed to have evolved from a nonenzymatic ancestor related to the widely distributed fatty-acid binding proteins (FAPs) and a plant protein family with no isomerase activity (CHILs). Ancestral inference supported the evolution of CHI from a protein lacking isomerase activity.

A coupled in vitro/in vivo approach for engineering a heterologous type III PKS to enhance polyketide biosynthesis in Saccharomyces cerevisiae.

Polyketides are attractive compounds for uses ranging from biorenewable chemical precursors to high-value therapeutics. In many cases, synthesis in a heterologous host is required to produce these compounds in industrially relevant quantities. The type III polyketide synthase 2-pyrone synthase (2-PS) from Gerbera hybrida was used for the production of triacetic acid lactone (TAL) in Saccharomyces cerevisiae.

Molecular architectures of benzoic acid-specific type III polyketide synthases.

Biphenyl synthase and benzophenone synthase constitute an evolutionarily distinct clade of type III polyketide synthases (PKSs) that use benzoic acid-derived substrates to produce defense metabolites in plants. The use of benzoyl-CoA as an endogenous substrate is unusual for type III PKSs. Moreover, sequence analyses indicate that the residues responsible for the functional diversification of type III PKSs are mutated in benzoic acid-specific type III PKSs.

A Red Algal Bourbonane Sesquiterpene Synthase Defined by Microgram-Scale NMR-Coupled Crystalline Sponge X-ray Diffraction Analysis.

Sesquiterpene scaffolds are the core backbones of many medicinally and industrially important natural products. A plethora of sesquiterpene synthases, widely present in bacteria, fungi, and plants, catalyze the formation of these intricate structures often with multiple stereocenters starting from linear farnesyl diphosphate substrates. Recent advances in next-generation sequencing and metabolomics technologies have greatly facilitated gene discovery for sesquiterpene synthases.

Structural basis for specific ligation of the peroxisome proliferator-activated receptor δ.

The peroxisome proliferator-activated receptor (PPAR) family comprises three subtypes: PPARα, PPARγ, and PPARδ. PPARδ transcriptionally modulates lipid metabolism and the control of energy homeostasis; therefore, PPARδ agonists are promising agents for treating a variety of metabolic disorders. In the present study, we develop a panel of rationally designed PPARδ agonists.