Glycyl Radical Enzymes and Sulfonate Metabolism in the Microbiome.

Sulfonates include diverse natural products and anthropogenic chemicals and are widespread in the environment. Many bacteria can degrade sulfonates and obtain sulfur, carbon, and energy for growth, playing important roles in the biogeochemical sulfur cycle. Cleavage of the inert sulfonate C-S bond involves a variety of enzymes, cofactors, and oxygen-dependent and oxygen-independent catalytic mechanisms. Sulfonate degradation by strictly anaerobic bacteria was recently found to involve C-S bond cleavage through O2-sensitive free radical chemistry, catalyzed by glycyl radical enzymes (GREs). The associated discoveries of new enzymes and metabolic pathways for sulfonate metabolism in diverse anaerobic bacteria have enriched our understanding of sulfonate chemistry in the anaerobic biosphere. An anaerobic environment of particular interest is the human gut microbiome, where sulfonate degradation by sulfate- and sulfite-reducing bacteria (SSRB) produces H2S, a process linked to certain chronic diseases and conditions.

The Human Knockout Gene CLYBL Connects Itaconate to Vitamin B12.

CLYBL encodes a ubiquitously expressed mitochondrial enzyme, conserved across all vertebrates, whose cellular activity and pathway assignment are unknown. Its homozygous loss is tolerated in seemingly healthy individuals, with reduced circulating B12 levels being the only and consistent phenotype reported to date. Here, by combining enzymology, structural biology, and activity-based metabolomics, we report that CLYBL operates as a citramalyl-CoA lyase in mammalian cells. Cells lacking CLYBL accumulate citramalyl-CoA, an intermediate in the C5-dicarboxylate metabolic pathway that includes itaconate, a recently identified human anti-microbial metabolite and immunomodulator. We report that CLYBL loss leads to a cell-autonomous defect in the mitochondrial B12 metabolism and that itaconyl-CoA is a cofactor-inactivating, substrate-analog inhibitor of the mitochondrial B12-dependent methylmalonyl-CoA mutase (MUT). Our work de-orphans the function of human CLYBL and reveals that a consequence of exposure to the immunomodulatory metabolite itaconate is B12 inactivation.

Second-Shell Amino Acid R266 Helps Determine N-Succinylamino Acid Racemase Reaction Specificity in Promiscuous N-Succinylamino Acid Racemase/o-Succinylbenzoate Synthase Enzymes.

Catalytic promiscuity is the coincidental ability to catalyze nonbiological reactions in the same active site as the native biological reaction. Several lines of evidence show that catalytic promiscuity plays a role in the evolution of new enzyme functions. Thus, studying catalytic promiscuity can help identify structural features that predispose an enzyme to evolve new functions. This study identifies a potentially preadaptive residue in a promiscuous N-succinylamino acid racemase/o-succinylbenzoate synthase (NSAR/OSBS) enzyme from Amycolatopsis sp. T-1-60. This enzyme belongs to a branch of the OSBS family which includes many catalytically promiscuous NSAR/OSBS enzymes. R266 is conserved in all members of the NSAR/OSBS subfamily. However, the homologous position is usually hydrophobic in other OSBS subfamilies, whose enzymes lack NSAR activity. The second-shell amino acid R266 is close to the catalytic acid/base K263, but it does not contact the substrate, suggesting that R266 could affect the catalytic mechanism. Mutating R266 to glutamine in Amycolatopsis NSAR/OSBS profoundly reduces NSAR activity but moderately reduces OSBS activity. This is due to a 1000-fold decrease in the rate of proton exchange between the substrate and the general acid/base catalyst K263. This mutation is less deleterious for the OSBS reaction because K263 forms a cation-π interaction with the OSBS substrate and/or the intermediate, rather than acting as a general acid/base catalyst. Together, the data explain how R266 contributes to NSAR reaction specificity and was likely an essential preadaptation for the evolution of NSAR activity.

Influence of Water and Enzyme on the Post-Transition State Bifurcation of NgnD-Catalyzed Ambimodal [6+4]/[4+2] Cycloaddition.

The enzyme NgnD catalyzes an ambimodal cycloaddition that bifurcates to [6+4]- and [4+2]-adducts. Both products have been isolated in experiments, but it remains unknown how enzyme and water influence the bifurcation selectivity at the femtosecond time scale. Here, we study the impact of water and enzyme on the post-transition state bifurcation of NgnD-catalyzed [6+4]/[4+2] cycloaddition by integrating quantum mechanics/molecular mechanics quasiclassical dynamics simulations and biochemical assays. The ratio of [6+4]/[4+2] products significantly differs in the gas phase, water, and enzyme. Biochemical assays were employed to validate computational predictions. The study informs how water and enzyme affect the bifurcation selectivity through perturbation of the reaction dynamics in the femtosecond time scale, revealing the fundamental roles of condensed media in dynamically controlling the chemical selectivity for biosynthetic reactions.

An Aromatic Cluster in the Active Site of epi-Isozizaene Synthase Is an Electrostatic Toggle for Divergent Terpene Cyclization Pathways.

The sesquiterpene cyclase epi-isozizaene synthase (EIZS) catalyzes the cyclization of farnesyl diphosphate to form the tricyclic precursor of the antibiotic albaflavenone. The hydrophobic active site is largely defined by aromatic residues that direct a multistep reaction sequence through multiple carbocation intermediates. The previous substitution of polar residues for a key aromatic residue, F96, converts EIZS into a high-fidelity sesquisabinene synthase: the F96S, F96M, and F96Q variants generate 78%, 91%, and 97% sesquisabinene A, respectively. Here, we report high-resolution X-ray crystal structures of two of these reprogrammed cyclases. The structures of the F96M EIZS-Mg2+3-risedronate and F96M EIZS-Mg2+3-inorganic pyrophosphate-benzyltriethylammonium cation complexes reveal structural changes in the F96 aromatic cluster that redirect the cyclization pathway leading from the bisabolyl carbocation intermediate in catalysis. The structure of the F96S EIZS-Mg2+3-neridronate complex reveals a partially occupied inhibitor and an enzyme active site caught in transition between open and closed states. Finally, three structures of wild-type EIZS complexed with the bisphosphonate inhibitors neridronate, pamidronate, and risedronate provide a foundation for understanding binding differences between wild-type and variant enzymes. These structures provide new insight regarding active site flexibility, particularly with regard to the potential for subtle expansion and contraction to accommodate ligands of varying sizes as well as bound water molecules. Additionally, these structures highlight the importance of conformational changes in the F96 aromatic cluster that could influence cation-π interactions with carbocation intermediates in catalysis.

Biochemical characterization of 2-phosphinomethylmalate synthase from Streptomyces hygroscopicus: A member of the DRE-TIM metallolyase superfamily.

2-Phosphinomethylmalate synthase (PMMS) from Streptomyces hygroscopicus catalyzes the first step in the biosynthesis of the herbicide bialophos using 3-phosphinopyruvic acid and acetyl coenzyme A as substrates to form 2-phosphinomethylmalic acid and coenzyme A. PMMS belongs to the Claisen condensation-like (CC-like) subgroup of the DRE-TIM metallolyase superfamily, which uses conserved active site architecture to catalyze a functionally-diverse set of reactions. Analysis of a sequence similarity network for the CC-like subgroup identified PMMS and the related R-citrate synthase in an early-diverging cluster suggesting that this group of sequences are more distinct in relation to other Claisen-condensation subgroup members. To better understand the structure/function landscape of the CC-like subgroup PMMS was recombinantly expressed in Escherichia coli, purified, and characterized with respect to its enzymatic properties. Using oxaloacetate as a substrate analog, the recombinantly-produced enzyme exhibited improved Michaelis constants relative to the previously reported natively-produced enzyme. Results from pH rate profiles and kinetic isotope effects were consistent with results from other members of the CC-like subgroup supporting acid-base chemistry and hydrolysis of the direct Claisen-condensation product as the rate-determining step. Results of site-directed mutagenesis experiments indicate that PMMS uses an active-site architecture similar to homocitrate synthase to select for a dicarboxylic acid substrate.

Accelerating Biphasic Biocatalysis through New Process Windows.

Process intensification through continuous flow reactions has increased the production rates of fine chemicals and pharmaceuticals. Catalytic reactions are accelerated through an unconventional and unprecedented use of a high-performance liquid/liquid counter current chromatography system. Product generation is significantly faster than in traditional batch reactors or in segmented flow systems, which is exemplified through stereoselective phase-transfer catalyzed reactions. This methodology also enables the intensification of biocatalysis as demonstrated in high yield esterifications and in the sesquiterpene cyclase-catalyzed synthesis of sesquiterpenes from farnesyl diphosphate as high-value natural products with applications in medicine, agriculture and the fragrance industry. Product release in sesquiterpene synthases is rate limiting due to the hydrophobic nature of sesquiterpenes, but a biphasic system exposed to centrifugal forces allows for highly efficient reactions.

Crystal structure of F95Q epi-isozizaene synthase, an engineered sesquiterpene cyclase that generates biofuel precursors ß- and γ-curcumene.

The saturated hydrocarbon bisabolane is a diesel fuel substitute that can be derived from sesquiterpene precursors bisabolene or curcumene. These sesquiterpenes are generated from farnesyl diphosphate in reactions catalyzed by eponymous terpenoid cyclases, but they can also be generated by engineered terpenoid cyclases in which cyclization cascades have been reprogrammed by mutagenesis. Here, we describe the X-ray crystal structure determination of F95Q epi-isozizaene synthase (EIZS), in which the new activity of curcumene biosynthesis has been introduced and the native activity of epi-isozizaene biosynthesis has been suppressed. F95Q EIZS generates ß- and γ-curcumene regioisomers with greater than 50% yield. Structural analysis of the closed active site conformation, stabilized by the binding of 3 Mg2+ ions, inorganic pyrophosphate, and the benzyltriethylammonium cation, reveals a product-like active site contour that serves as the cyclization template. Remolding the active site contour to resemble curcumene instead of epi-isozizaene is the principal determinant of the reprogrammed cyclization cascade. Intriguingly, an ordered water molecule comprises part of the active site contour. This water molecule may also serve as a final proton acceptor, along with inorganic pyrophosphate, in the generation of curcumene regioisomers; it may also contribute to the formation of sesquiterpene alcohols identified as minor side products. Thus, the substitution of polar side chains for nonpolar side chains in terpenoid cyclase active sites can result in the stabilization of bound water molecules that, in turn, can serve template functions in isoprenoid cyclization reactions.

Genome-Mined Diels-Alderase Catalyzes Formation of the cis-Octahydrodecalins of Varicidin A and B.

Pericyclases are an emerging family of enzymes catalyzing pericyclic reactions. A class of lipocalin-like enzymes recently characterized as Diels-Alderases (DAses) catalyze decalin formation through intramolecular Diels-Alder (IMDA) reactions between electron-rich dienes and electron-deficient dienophiles. Using this class of enzyme as a beacon for genome mining, we discovered a biosynthetic gene cluster from Penicillium variabile and identified that it encodes for the biosynthesis of varicidin A (1), a new antifungal natural product containing a cis-octahydrodecalin core. Biochemical analysis reveals a carboxylative deactivation strategy used in varicidin biosynthesis to suppress the nonenzymatic IMDA reaction of an early acyclic intermediate that favors trans-decalin formation. A P450 oxidizes the reactive intermediate to yield a relatively unreactive combination of an electron-deficient diene and an electron-deficient dienophile. The DAse PvhB catalyzes the final stage IMDA on the carboxylated intermediate to form the cis-decalin that is important for the antifungal activity.

Sesquiterpenoids Produced by Combining Two Sesquiterpene Cyclases with Promiscuous Myxobacterial CYP260B1.

Sesquiterpenes represent a class of important terpenoids with high structural diversity and a wide range of applications. The cyclized core skeletons are generated by sesquiterpene cyclases, and the structural diversity is further increased by a series of modification steps. Cytochromes P450 (P450s) are a class of monooxygenases and one of the main contributors to the structural diversity of natural products. Some of these P450s show a broad substrate range and might be promising candidates for the implementation of cascade reactions. In this study, a combinatorial biosynthesis approach was utilized by the combination of a promiscuous myxobacterial P450 (CYP260B1) with two sesquiterpene cyclases (FgJ01056, FgJ09920) of filamentous fungi. Two oxygenated products, culmorin and culmorone, and a new compound, koraidiol, were successfully generated and characterized. This approach suggests the potential use of noncognate P450s to produce novel oxygenated terpenoids, or to generate a novel biosynthetic route for known terpenoids by a combinatorial biosynthesis strategy.

Structural and Chemical Biology of Terpenoid Cyclases.

The year 2017 marks the twentieth anniversary of terpenoid cyclase structural biology: a trio of terpenoid cyclase structures reported together in 1997 were the first to set the foundation for understanding the enzymes largely responsible for the exquisite chemodiversity of more than 80000 terpenoid natural products. Terpenoid cyclases catalyze the most complex chemical reactions in biology, in that more than half of the substrate carbon atoms undergo changes in bonding and hybridization during a single enzyme-catalyzed cyclization reaction. The past two decades have witnessed structural, functional, and computational studies illuminating the modes of substrate activation that initiate the cyclization cascade, the management and manipulation of high-energy carbocation intermediates that propagate the cyclization cascade, and the chemical strategies that terminate the cyclization cascade. The role of the terpenoid cyclase as a template for catalysis is paramount to its function, and protein engineering can be used to reprogram the cyclization cascade to generate alternative and commercially important products. Here, I review key advances in terpenoid cyclase structural and chemical biology, focusing mainly on terpenoid cyclases and related prenyltransferases for which X-ray crystal structures have informed and advanced our understanding of enzyme structure and function.

An Unusual Skeletal Rearrangement in the Biosynthesis of the Sesquiterpene Trichobrasilenol from Trichoderma.

The skeletons of some classes of terpenoids are unusual in that they contain a larger number of Me groups (or their biosynthetic equivalents such as olefinic methylene groups, hydroxymethyl groups, aldehydes, or carboxylic acids and their derivatives) than provided by their oligoprenyl diphosphate precursor. This is sometimes the result of an oxidative ring-opening reaction at a terpene-cyclase-derived molecule containing the regular number of Me group equivalents, as observed for picrotoxan sesquiterpenes. In this study a sesquiterpene cyclase from Trichoderma spp. is described that can convert farnesyl diphosphate (FPP) directly via a remarkable skeletal rearrangement into trichobrasilenol, a new brasilane sesquiterpene with one additional Me group equivalent compared to FPP. A mechanistic hypothesis for the formation of the brasilane skeleton is supported by extensive isotopic labelling studies.

Crystal Structure of Cucumene Synthase, a Terpenoid Cyclase That Generates a Linear Triquinane Sesquiterpene.

Linear triquinanes are sesquiterpene natural products with hydrocarbon skeletons consisting of three fused five-membered rings. Importantly, several of these compounds exhibit useful anticancer, anti-inflammatory, and antibiotic properties. However, linear triquinanes pose significant challenges to organic synthesis because of the structural and stereochemical complexity of their hydrocarbon skeletons. To illuminate nature's solution to the generation of linear triquinanes, we now describe the crystal structure of Streptomyces clavuligerus cucumene synthase. This sesquiterpene cyclase catalyzes the stereospecific cyclization of farnesyl diphosphate to form a linear triquinane product, (5 S,7 S,10 R,11 S)-cucumene. Specifically, we report the structure of the wild-type enzyme at 3.05 Å resolution and the structure of the T181N variant at 1.96 Å resolution, both in the open active site conformations without any bound ligands. The high-resolution structure of T181N cucumene synthase enables inspection of the active site contour, which adopts a three-dimensional shape complementary to a linear triquinane. Several aromatic residues outline the active site contour and are believed to facilitate cation-π interactions that would stabilize carbocation intermediates in catalysis. Thus, aromatic residues in the active site not only define the template for catalysis but also play a role in reducing activation barriers in the multistep cyclization cascade.

Comparison of Alicyclobacillus acidocaldarius o-Succinylbenzoate Synthase to Its Promiscuous N-Succinylamino Acid Racemase/ o-Succinylbenzoate Synthase Relatives.

Studying the evolution of catalytically promiscuous enzymes like those from the N-succinylamino acid racemase/ o-succinylbenzoate synthase (NSAR/OSBS) subfamily can reveal mechanisms by which new functions evolve. Some enzymes in this subfamily have only OSBS activity, while others catalyze OSBS and NSAR reactions. We characterized several NSAR/OSBS subfamily enzymes as a step toward determining the structural basis for evolving NSAR activity. Three enzymes were promiscuous, like most other characterized NSAR/OSBS subfamily enzymes. However, Alicyclobacillus acidocaldarius OSBS (AaOSBS) efficiently catalyzes OSBS activity but lacks detectable NSAR activity. Competitive inhibition and molecular modeling show that AaOSBS binds N-succinylphenylglycine with moderate affinity in a site that overlaps its normal substrate. On the basis of possible steric conflicts identified by molecular modeling and sequence conservation within the NSAR/OSBS subfamily, we identified one mutation, Y299I, that increased NSAR activity from undetectable to 1.2 × 102 M-1 s-1 without affecting OSBS activity. This mutation does not appear to affect binding affinity but instead affects kcat, by reorienting the substrate or modifying conformational changes to allow both catalytic lysines to access the proton that is moved during the reaction. This is the first site known to affect reaction specificity in the NSAR/OSBS subfamily. However, this gain of activity was obliterated by a second mutation, M18F. Epistatic interference by M18F was unexpected because a phenylalanine at this position is important in another NSAR/OSBS enzyme. Together, modest NSAR activity of Y299I AaOSBS and epistasis between sites 18 and 299 indicate that additional sites influenced the evolution of NSAR reaction specificity in the NSAR/OSBS subfamily.

A Radical Intermediate in Bacillus subtilis QueE during Turnover with the Substrate Analogue 6-Carboxypterin.

7-Carboxy-7-deazaguanine (CDG) synthase (QueE), a member of the radical S-deoxyadenosyl-l-methionine (SAM) superfamily of enzymes, catalyzes a radical-mediated ring rearrangement required to convert 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) into CDG, forming the 7-dezapurine precursor to all pyrrolopyrimidine metabolites. Members of the radical SAM superfamily bind SAM to a [4Fe-4S] cluster, leveraging the reductive cleavage of SAM by the cluster to produce a highly reactive 5'-deoxyadenosyl radical which initiates chemistry by H atom abstraction from the substrate. QueE has recently been shown to use 6-carboxypterin (6-CP) as an alternative substrate, forming 6-deoxyadenosylpterin as the product. This reaction has been proposed to occur by radical addition between 5'-dAdo· and 6-CP, which upon oxidative decarboxylation yields the modified pterin. Here, we present spectroscopic evidence for a 6-CP-dAdo radical. The structure of this intermediate is determined by characterizing its electronic structure by continuous wave and pulse electron paramagnetic resonance spectroscopy.

Human Cytochrome CYP17A1: The Structural Basis for Compromised Lyase Activity with 17-Hydroxyprogesterone.

The multifunctional enzyme, cytochrome P450 (CYP17A1), plays a crucial role in the production of androgens, catalyzing two key reactions on pregnenolone (PREG) and progesterone (PROG), the first being a 17-hydroxylation to generate 17-OH PREG and 17-OH PROG, with roughly equal efficiencies. The second is a C-C bond scission or "lyase" reaction in which the C17-C20 bond is cleaved, leading to the eventual production of powerful androgens, whose involvement in the proliferation of prostate cancer has generated intense interest in developing inhibitors of CYP17A1. For humans, the significance of the C-C bond cleavage of 17-OH PROG is lessened, because it is about 50 times less efficient than for 17-OH PREG in terms of kcat/Km. Recognizing the need to clarify relevant reaction mechanisms involved with such transformations, we first report studies of solvent isotope effects, results of which are consistent with a Compound I mediated PROG hydroxylase activity, yet exclude this intermediate as a participant in the formation of androstenedione (AD) via the lyase reaction. This finding is also supported by a combination of cryoreduction and resonance Raman spectroscopy that traps and structurally characterizes the key hemiketal reaction intermediates. Adding to a previous study of PREG and 17-OH PREG metabolism, the current work provides definitive evidence for a more facile protonation of the initially formed ferric peroxo-intermediate for 17-OH PROG-bound CYP17A1, compared to the complex with 17-OH PREG. Importantly, Raman characterization also reveals an H-bonding interaction with the terminal oxygen of the peroxo fragment, rather than with the proximal oxygen, as is present for 17-OH PREG. These factors would favor a diminished lyase activity of the sample with 17-OH PROG relative to the complex with 17-OH PREG, thereby providing a convincing structural explanation for the dramatic differences in activity for these lyase substrates in humans.

Radical S-Adenosyl-l-methionine Tryptophan Lyase (NosL): How the Protein Controls the Carboxyl Radical •CO2- Migration.

The radical S-adenosyl-l-methionine tryptophan lyase uses radical-based chemistry to convert l-tryptophan into 3-methyl-2-indolic acid, a fragment in the biosynthesis of the thiopeptide antibiotic nosiheptide. This complex reaction involves several successive steps corresponding to (i) the activation by a specific hydrogen-atom abstraction, (ii) an unprecedented •CO2- radical migration, (iii) a cyanide fragment release, and (iv) the termination of the radical-based reaction. In vitro study of this reaction is made more difficult because the enzyme produces a significant amount of a shunt product instead of the natural product. Here, using a combination of X-ray crystallography, electron paramagnetic resonance spectroscopy, and quantum and hybrid quantum mechanical/molecular mechanical calculations, we have deciphered the fine mechanism of the key •CO2- radical migration, highlighting how the preorganized active site of the protein tightly controls this reaction.

Following the electrons: peculiarities in the catalytic cycles of radical SAM enzymes.

Radical SAM enzymes use S-adenosyl-l-methionine as an oxidant to initiate radical-mediated transformations that would otherwise not be possible with Lewis acid/base chemistry alone. These reactions are either redox neutral or oxidative leading to certain expectations regarding the role of SAM as either a reusable cofactor or the ultimate electron acceptor during each turnover. However, these expectations are frequently not realized resulting in fundamental questions regarding the redox handling and movement of electrons associated with these biological catalysts. Herein we provide a focused perspective on several of these questions and associated hypotheses with an emphasis on recently discovered radical SAM enzymes.

Light-Activated Electron Transfer and Turnover in Ru-Modified Aldehyde Deformylating Oxygenases.

Conversion of biological molecules into fuels or other useful chemicals is an ongoing chemical challenge. One class of enzymes that has received attention for such applications is aldehyde deformylating oxygenase (ADO) enzymes. These enzymes convert aliphatic aldehydes to the alkanes and formate. In this work, we prepared and investigated ADO enzymes modified with RuII(tris-diimine) photosensitizers as a starting point for probing intramolecular electron transfer events. Three variants were prepared, with RuII-modification at the wild type (WT) residue C70, at the R62C site in one mutant ADO, and at both C62 and C70 in a second mutant ADO protein. The single-site modification of WT ADO at C70 using a cysteine-reactive label is an important observation and opens a way forward for new studies of electron flow, mechanism, and redox catalysis in ADO. These Ru-ADO constructs can perform the ADO catalytic cycle in the presence of light and a sacrificial reductant. In this work, the Ru photosensitizer serves as a tethered, artificial reductase that promotes turnover of aldehyde substrates with different carbon chain lengths. Peroxide side products were detected for shorter chain aldehydes, concomitant with less productive turnover. Analysis using semiclassical electron transfer theory supports proposals for hopping pathway for electron flow in WT ADO and in our new Ru-ADO proteins.

Biocatalysis with the milk protein ß-lactoglobulin: promoting retroaldol cleavage of α,ß-unsaturated aldehydes.

Enzymes with a hydrophobic binding site and an active site lysine have been suggested to be promiscuous in their catalytic activity. ß-Lactoglobulin (BLG), the principle whey protein found in milk, possesses a central calyx that binds non-polar molecules. Here, we report that BLG can catalyze the retro-aldol cleavage of α,ß-unsaturated aldehydes making it a naturally occurring protein capable of catalyzing retro-aldol reactions on hydrophobic substrates. Retroaldolase activity was seen to be most effective on substrates with phenyl or naphthyl side-chains. Use of a brominated substrate analogue inhibitor increases the product yield by a factor of three. BLG's catalytic activity and its ready availability make it a prime candidate for the development of commercial biocatalysts.