Changing Fates of the Substrate Radicals Generated in the Active Sites of the B12-Dependent Radical SAM Enzymes OxsB and AlsB.

OxsB is a B12-dependent radical SAM enzyme that catalyzes the oxidative ring contraction of 2'-deoxyadenosine 5'-phosphate to the dehydrogenated, oxetane containing precursor of oxetanocin A phosphate. AlsB is a homologue of OxsB that participates in a similar reaction during the biosynthesis of albucidin. Herein, OxsB and AlsB are shown to also catalyze radical mediated, stereoselective C2'-methylation of 2'-deoxyadenosine monophosphate. This reaction proceeds with inversion of configuration such that the resulting product also possesses a C2' hydrogen atom available for abstraction. However, in contrast to methylation, subsequent rounds of catalysis result in C-C dehydrogenation of the newly added methyl group to yield a 2'-methylidene followed by radical addition of a 5'-deoxyadenosyl moiety to produce a heterodimer. These observations expand the scope of reactions catalyzed by B12-dependent radical SAM enzymes and emphasize the susceptibility of radical intermediates to bifurcation along different reaction pathways even within the highly organized active site of an enzyme.

Flavin-enabled reductive and oxidative epoxide ring opening reactions.

Epoxide ring opening reactions are common and important in both biological processes and synthetic applications and can be catalyzed in a non-redox manner by epoxide hydrolases or reductively by oxidoreductases. Here we report that fluostatins (FSTs), a family of atypical angucyclines with a benzofluorene core, can undergo nonenzyme-catalyzed epoxide ring opening reactions in the presence of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). The 2,3-epoxide ring in FST C is shown to open reductively via a putative enol intermediate, or oxidatively via a peroxylated intermediate with molecular oxygen as the oxidant. These reactions lead to multiple products with different redox states that possess a single hydroxyl group at C-2, a 2,3-vicinal diol, a contracted five-membered A-ring, or an expanded seven-membered A-ring. Similar reactions also take place in both natural products and other organic compounds harboring an epoxide adjacent to a carbonyl group that is conjugated to an aromatic moiety. Our findings extend the repertoire of known flavin chemistry that may provide new and useful tools for organic synthesis.

Byproduct formation during the biosynthesis of spinosyn A and evidence for an enzymatic interplay to prevent its formation.

Biosynthesis of spinosyn A in Saccharopolyspora spinosa involves a 1,4-dehydration followed by an intramolecular [4 + 2]-cycloaddition catalyzed by SpnM and SpnF, respectively. The cycloaddition also takes place in the absence of SpnF leading to questions regarding its mechanism of catalysis and biosynthetic role. Substrate analogs were prepared with an unactivated dienophile or an acyclic structure and found to be unreactive consistent with the importance of these features for cyclization. The SpnM-catalyzed dehydration reaction was also found to yield a byproduct corresponding to the C11 = C12 cis isomer of the SpnF substrate. This byproduct is stable both in the presence and absence of SpnF; however, relative production of the SpnM product and byproduct could be shifted in favor of the former by including SpnF or the dehydrogenase SpnJ in the reaction. This result suggests a potential interplay between the enzymes of spinosyn A biosynthesis that may help to improve the efficiency of the pathway.

Dioxane Bridge Formation during the Biosynthesis of Spectinomycin Involves a Twitch Radical S-Adenosyl Methionine Dehydrogenase That May Have Evolved from an Epimerase.

Spectinomycin is a dioxane-bridged, tricyclic aminoglycoside produced by Streptomyces spectabilis ATCC 27741. While the spe biosynthetic gene cluster for spectinomycin has been reported, the chemistry underlying construction of the dioxane ring is unknown. The twitch radical SAM enzyme SpeY from the spe cluster is shown here to catalyze dehydrogenation of the C2' alcohol of (2'R,3'S)-tetrahydrospectinomycin to yield (3'S)-dihydrospectinomycin as a likely biosynthetic intermediate. This reaction is radical-mediated and initiated via H atom abstraction from C2' of the substrate by the 5'-deoxyadenosyl radical equivalent generated upon reductive cleavage of SAM. Crystallographic analysis of the ternary Michaelis complex places serine-183 adjacent to C2' of the bound substrate opposite C5' of SAM. Mutation of this residue to cysteine converts SpeY to the corresponding C2' epimerase mirroring the opposite phenomenon observed in the homologous twitch radical SAM epimerase HygY from the hygromycin B biosynthetic pathway. Phylogenetic analysis suggests a relatively recent evolutionary branching of putative twitch radical SAM epimerases bearing homologous cysteine residues to generate the SpeY clade of enzymes.

Radical S-Adenosyl Methionine Enzyme BlsE Catalyzes a Radical-Mediated 1,2-Diol Dehydration during the Biosynthesis of Blasticidin S.

The biosynthesis of blasticidin S has drawn attention due to the participation of the radical S-adenosyl methionine (SAM) enzyme BlsE. The original assignment of BlsE as a radical-mediated, redox-neutral decarboxylase is unusual because this reaction appears to serve no biosynthetic purpose and would need to be reversed by a subsequent carboxylation step. Furthermore, with the exception of BlsE, all other radical SAM decarboxylases reported to date are oxidative in nature. Careful analysis of the BlsE reaction, however, demonstrates that BlsE is not a decarboxylase but instead a lyase that catalyzes the dehydration of cytosylglucuronic acid (CGA) to form cytosyl-4'-keto-3'-deoxy-d-glucuronic acid, which can rapidly decarboxylate nonenzymatically in vitro. Analysis of substrate isotopologs, fluorinated analogues, as well as computational models based on X-ray crystal structures of the BlsE·SAM (2.09 Å) and BlsE·SAM·CGA (2.62 Å) complexes suggests that BlsE catalysis likely proceeds via direct elimination of water from the CGA C4' α-hydroxyalkyl radical as opposed to 1,2-migration of the C3'-hydroxyl prior to dehydration. Biosynthetic and mechanistic implications of the revised assignment of BlsE are discussed.

Evidence for an Enzyme-Catalyzed Rauhut-Currier Reaction during the Biosynthesis of Spinosyn A.

The catalog of enzymes known to catalyze the nucleophile-assisted formation of C-C bonds is extremely small, and there is presently no definitive example of a biological Rauhut-Currier reaction. Biosynthesis of the polyketide insecticide spinosyn A in Saccharopolyspora spinosa involves a [4 + 2]-cycloaddition and a subsequent intramolecular C-C bond formation catalyzed by SpnF and SpnL, respectively. Isotope tracer experiments and kinetic isotope effects, however, imply that the SpnL-catalyzed reaction proceeds without initial deprotonation of the substrate. The crystal structure of SpnL exhibits high similarity to SAM-dependent methyltransferases as well as SpnF. The residue Cys60 is also shown to reside in the SpnL active site, and the Cys60Ala SpnL mutant is found to be devoid of activity. Moreover, SpnL is covalently modified at Cys60 and irreversibly inactivated when it is coincubated with a fluorinated substrate analogue designed as a suicide inactivator of nucleophile-assisted C-C bond formation. These results suggest that SpnL catalyzes a biological Rauhut-Currier reaction.

Characterization of the coformycin biosynthetic gene cluster in Streptomyces kaniharaensis.

Coformycin and pentostatin are structurally related N-nucleoside inhibitors of adenosine deaminase characterized by an unusual 1,3-diazepine nucleobase. Herein, the cof gene cluster responsible for coformycin biosynthesis is identified. Reconstitution of the coformycin biosynthetic pathway in vitro demonstrates that it overlaps significantly with the early stages of l-histidine biosynthesis. Committed entry into the coformycin pathway takes place via conversion of a shared branch point intermediate to 8-ketocoformycin-[Formula: see text]-monophosphate catalyzed by CofB, which is a homolog of succinylaminoimidazolecarboxamide ribotide (SAICAR) synthetase. This reaction appears to proceed via a Dieckmann cyclization and a retro-aldol elimination, releasing ammonia and D-erythronate-4-phosphate as coproducts. Completion of coformycin biosynthesis involves reduction and dephosphorylation of the CofB product, with the former reaction being catalyzed by the NADPH-dependent dehydrogenase CofA. CofB also shows activation by adenosine triphosphate (ATP) despite the reaction requiring neither a phosphorylated nor an adenylated intermediate. This may serve to help regulate metabolic partitioning between the l-histidine and coformycin pathways.

Measurement of Net Rate Constants from Enzyme Progress Curves without Curve Fitting.

A method is described whereby net rate constants can be directly inferred from the progress curves of enzyme intermediates without the need for model specification, numerical analysis, curve fitting, or the steady-state approximation. Specifically, if an enzyme intermediate in an ultimately irreversible serial subsequence is perturbed from and returns back to its equilibrium state as the substrate is consumed, then its net rate constant is given by the ratio of the total substrate consumed and the area under the progress curve for the enzyme intermediate. A rigorous analysis demonstrates this result to hold independent of the complete enzymatic reaction in which the subsequence is embedded, making it broadly applicable to a very wide range of kinetic mechanisms, including those complicated by inhibition. As a theoretical consequence, it is shown that traditionally steady-state parameters such as kcat, kcat/KM, and net rate constants can be expressed as limiting ratios of averages without requiring the steady-state hypothesis. Finally, a mock data set is generated for a system of contemporary interest that can serve as both an example of how the methodology would be used in practice and a proof of concept.

Changes in Regioselectivity of H Atom Abstraction during the Hydroxylation and Cyclization Reactions Catalyzed by Hyoscyamine 6ß-Hydroxylase.

Hyoscyamine 6ß-hydroxylase (H6H) is an αKG-dependent nonheme iron oxidase that catalyzes the oxidation of hyoscyamine to scopolamine via two separate reactions: hydroxylation followed by oxidative cyclization. Both of these reactions are expected to involve H atom abstraction from each of two adjacent carbon centers (C6 vs C7) in the substrate. During hydroxylation, there is a roughly 85:1 preference for H atom abstraction from C6 versus C7; however, this inverts to a 1:16 preference during cyclization. Furthermore, 18O incorporation experiments in the presence of deuterated substrate are consistent with the catalytic iron(IV)-oxo complex being able to support the coordination of an additional ligand during hydroxylation. These observations suggest that subtle differences in the substrate binding configuration can have significant consequences for the catalytic cycle of H6H.

Substrate Conformation Correlates with the Outcome of Hyoscyamine 6ß-Hydroxylase Catalyzed Oxidation Reactions.

Hyoscyamine 6ß-hydroxylase (H6H) is an α-ketoglutarate dependent mononuclear nonheme iron enzyme that catalyzes C6-hydroxylation of hyoscyamine and oxidative cyclization of the resulting product to give the oxirane natural product scopolamine. Herein, the chemistry of H6H is investigated using hyoscyamine derivatives with modifications at the C6 or C7 position as well as substrate analogues possessing a 9-azabicyclo[3.3.1]nonane core. Results indicate that hydroxyl rebound is unlikely to take place during the cyclization reaction and that the hydroxylase versus oxidative cyclase activity of H6H is correlated with the presence of an exo-hydroxy group having syn-periplanar geometry with respect to the adjacent H atom to be abstracted.

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.

Mechanistic Implications of the Deamination of TDP-4-amino-4-deoxy-d-fucose Catalyzed by the Radical SAM Enzyme DesII.

DesII is a radical SAM lyase that catalyzes a deamination reaction during the biosynthesis of desosamine in Streptomyces venezuelae. Competing mechanistic hypotheses for this radical-mediated reaction are differentiated according to whether a 1,2-migration takes place and the timing of proton abstraction following generation of a substrate α-hydroxyalkyl radical intermediate. In this study, the deuterated C4 epimer of the natural substrate, TDP-4-amino-4-deoxy-d-[3-2H]fucose, was prepared and shown to be a substrate for DesII undergoing deamination alone with a specific activity that is only marginally reduced (∼3-fold) with respect to that of deamination of the natural substrate. Furthermore, pH titration of the deamination reaction implicates the presence of a hydron acceptor that facilitates catalysis but does not appear to be necessary. On the basis of these as well as previously reported results, a mechanism involving direct elimination of ammonium with concerted proton transfer to the nucleofuge from the adjacent α-hydroxyalkyl radical is proposed.

Theory and Application of the Relationship Between Steady-State Isotope Effects on Enzyme Intermediate Concentrations and Net Rate Constants.

Steady-state kinetic isotope effects on enzyme-catalyzed reactions are often interpreted in terms of the microscopic rate constants associated with the elementary reactions of interest. Unfortunately, this approach can lead to confusion, especially when more than one elementary reaction is isotopically sensitive, because it forces one to consider the full catalytic cycle one step at a time rather than as a complete whole. Herein we argue that shifting focus from intrinsic effects to net rate constants and enzyme intermediate concentrations provides a more natural and holistic interpretation by which the effects of partial rate limitation are more easily understood. In doing so, we demonstrate how the experimental determination of isotope effects on enzyme intermediate concentrations allows a direct determination of isotope effects on net rate constants. The chapter is divided into three main sections. The first outlines the basic theory and its interpretation. The second discusses an application of the theory in the study of the radical SAM enzyme DesII. The final section then provides the complete mathematical treatment.

Investigation of the mechanism of the SpnF-catalyzed [4+2]-cycloaddition reaction in the biosynthesis of spinosyn A.

The Diels-Alder reaction is one of the most common methods to chemically synthesize a six-membered carbocycle. While it has long been speculated that the cyclohexene moiety found in many secondary metabolites is also introduced via similar chemistry, the enzyme SpnF involved in the biosynthesis of the insecticide spinosyn A in Saccharopolyspora spinosa is the first enzyme for which catalysis of an intramolecular [Formula: see text]-cycloaddition has been experimentally verified as its only known function. Since its discovery, a number of additional standalone [Formula: see text]-cyclases have been reported as potential Diels-Alderases; however, whether their catalytic cycles involve a concerted or stepwise cyclization mechanism has not been addressed experimentally. Here, we report direct experimental interrogation of the reaction coordinate for the [Formula: see text]-carbocyclase SpnF via the measurement of [Formula: see text]-secondary deuterium kinetic isotope effects (KIEs) at all sites of [Formula: see text] rehybridization for both the nonenzymatic and enzyme-catalyzed cyclization of the SpnF substrate. The measured KIEs for the nonenzymatic reaction are consistent with previous computational results implicating an intermediary state between formation of the first and second carbon-carbon bonds. The KIEs measured for the enzymatic reaction suggest a similar mechanism of cyclization within the enzyme active site; however, there is evidence that conformational restriction of the substrate may play a role in catalysis.

Natural [4 + 2]-Cyclases.

[4 + 2]-Cycloadditions are increasingly being recognized in the biosynthetic pathways of many structurally complex natural products. A relatively small collection of enzymes from these pathways have been demonstrated to increase rates of cyclization and impose stereochemical constraints on the reactions. While mechanistic investigation of these enzymes is just beginning, recent studies have provided new insights with implications for understanding their biosynthetic roles, mechanisms of catalysis, and evolutionary origin.

Identification and Characterization of Enzymes Catalyzing Pyrazolopyrimidine Formation in the Biosynthesis of Formycin A.

Genome scanning of Streptomyces kaniharaensis, the producer of formycin A, reveals two sets of purA, purB, purC, and purH genes. The Pur enzymes catalyze pyrimidine assembly of purine nucleobases. To test whether enzymes encoded by the second set of pur genes catalyze analogous transformations in formycin biosynthesis, formycin B 5'-phosphate was synthesized and shown to be converted by ForA and ForB to formycin A 5'-phosphate. These results support that For enzymes are responsible for formycin formation.

Studies of 1-Amino-2,2-difluorocyclopropane-1-carboxylic Acid: Mechanism of Decomposition and Inhibition of 1-Aminocyclopropane-1-carboxylic Acid Deaminase.

1-Amino-2,2-difluorocyclopropane-1-carboxylic acid (DFACC) is of interest in the study of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase due to the increased reactivity of its cyclopropyl functionality. It is shown that DFACC is unstable under near-physiological conditions where it primarily decomposes via specific-base catalysis to 3-fluoro-2-oxobut-3-enoic acid with a rate constant of 0.18 ± 0.01 min(-1). Upon incubation with ACC deaminase, DFACC is found to be a slow-dissociating inhibitor of ACC deaminase with submicromolar affinity.

Mechanistic Investigation of the Radical S-Adenosyl-L-methionine Enzyme DesII Using Fluorinated Analogues.

DesII is a radical S-adenosyl-l-methionine (SAM) enzyme that can act as a deaminase or a dehydrogenase depending on the nature of its TDP-sugar substrate. Previous work has implicated a substrate-derived, C3-centered α-hydroxyalkyl radical as a key intermediate during catalysis. Although deprotonation of the α-hydroxyalkyl radical has been shown to be important for dehydrogenation, much less is known regarding the course of the deamination reaction. To investigate the role played by the C3 hydroxyl during deamination, 3-deutero-3-fluoro analogues of both substrates were prepared and characterized with DesII. In neither case was deamination or oxidation observed; however, in both cases deuterium was efficiently exchanged between the substrate analogues and SAM. These results imply that the C3 hydroxyl plays a key role in both reactions­thereby arguing against a 1,2-migration mechanism of deamination­and that homolysis of SAM concomitant with H atom abstraction from the substrate is readily reversible when forward partitioning is inhibited.