Divergent function in the crotonase superfamily: an anhydride intermediate in the reaction catalyzed by 3-hydroxyisobutyryl-CoA hydrolase

3-Hydroxyisobutyryl-CoA hydrolase (HICH), a member of the enoyl-CoA (crotonase) superfamily, catalyzes the hydrolysis of 3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate. Like other members of the superfamily, the sequence of HICH contains conserved sequences for an oxyanion hole that stabilizes anionic intermediates. In contrast to most members of the superfamily, the reaction catalyzed by HICH does not proceed via formation of a thioester enolate anion; instead, evidence based on substrate deuterium isotope effects, the reactivity of substrate analogues that cannot form thioester enolate anions, single-turnover experiments in H218O, and the kinetic phenotypes of site-directed mutants provide evidence for a mechanism involving the formation of an anhydride intermediate involving Glu143 in the active site. In the reactions catalyzed by many members of the superfamily, homologues of Glu143 abstract the alpha proton of the thioester substrate to generate the thioester enolate anion intermediate. Presumably, one or more of the anionic tetrahedral intermediates on the HICH reaction coordinate are stabilized by the oxyanion hole. Thus, we conclude that the conserved oxyanion hole in this superfamily can be used to stabilize a variety of anionic intermediates.     

In vitro reconstitution of α-pyrone ring formation in myxopyronin biosynthesis

Myxopyronins are α-pyrone antibiotics produced by the terrestrial bacterium Myxococcus fulvus Mx f50 and possess antibacterial activity against Gram-positive and Gram-negative pathogens. They target the bacterial RNA polymerase (RNAP) “switch region” as non-competitive inhibitors and display no cross-resistance to the established RNAP inhibitor rifampicin. Recent analysis of the myxopyronin biosynthetic pathway led to the hypothesis that this secondary metabolite is produced from two separate polyketide parts, which are condensed by the stand-alone ketosynthase MxnB. Using in vitro assays we show that MxnB catalyzes a unique condensation reaction forming the α-pyrone ring of myxopyronins from two activated acyl chains in form of their β-keto intermediates. MxnB is able to accept thioester substrates coupled to either N-acetylcysteamine (NAC) or a specific carrier protein (CP). The turnover rate of MxnB for substrates bound to CP was 12-fold higher than for NAC substrates, demonstrating the importance of protein–protein interactions in polyketide synthase (PKS) systems. The crystal structure of MxnB reveals the enzyme to be an unusual member of the ketosynthase group capable of binding and condensing two long alkyl chains bound to carrier proteins. The geometry of the two binding tunnels supports the biochemical data and allows us to propose an order of reaction, which is supported by the identification of novel myxopyronin congeners in the extract of the producer strain. Insights into the mechanism of this unique condensation reaction do not only expand our knowledge regarding the thiolase enzyme family but also opens up opportunities for PKS bioengineering to achieve directed structural modifications.     

The iterative gramicidin s thioesterase catalyzes peptide ligation and cyclization

Here, we present a comprehensive in vitro characterization of the excised iterative, bimodular PCP-TE of the gramicidin S synthetase GrsB, which is able to act both as a ligation and a cyclization catalyst. Using the native pentapeptidyl-thioester substrates, GrsB PCP-TE catalyzes the dimerization and subsequent formation of the decapeptide lactam gramicidin S. Interestingly, the detection of linear decapeptidyl-SNAC as an enzyme-dependent intermediate supports the iterative mechanism in vivo, in which two pentapeptides, one bound as an ester to the active site serine of the TE domain and the second bound as a thioester to the adjacent pan-PCP, are ligated to a decapeptidyl-pan-PCP that subsequently transferred to the adjacent TE domain and cyclized. Moreover, GrsB PCP-TE can handle different substrates length, leading not only to dimerization, but also to trimerization and the formation of different ring sizes.     

Simultaneous detection of cholyl adenylate and coenzyme A thioester utilizing liquid chromatography/electrospray ionization mass spectrometry.

It has been proposed that acyl adenylate is first formed during activation of the carboxy group into the acyl CoA thioester, an intermediate in the formation of amino acid conjugates. Acyl CoA synthetases may be responsible for this acyl adenylate formation. Recently, we hypothesized the preferential formation of cholic acid adenylate, a major bile acid, preceding production of the corresponding CoA thioester in incubations with rat liver microsomal fractions. To verify this biosynthetic mechanism, monitoring of the incubation mixture of acyl adenylate together with both substrate and acyl CoA thioester is needed. We have developed a detection method for the simultaneous detection of these cholic acid derivatives utilizing liquid chromatography/electrospray ionization mass spectrometry. The CoA thioester of cholic acid forms a chelation complex with the divalent cations remaining on the silica gel packed into the analytical column. Both the addition of a chelating agent, such as EDTA, to the mobile phase and an adjustment of the mobile phase pH to a weak alkaline effectively removed such chelate formation, producing a sharp CoA thioester peak. For a simultaneous mass spectrometric analysis of cholic acid, the corresponding adenylate and CoA thioester, the combined use of a 300 A particle diameter ODS column and 20 mM ammonium acetate buffer (pH 9.0)/2-propanol/acetonitrile as the mobile phase have been proved to be preferable. To avoid any degradation of the chemically unstable adenylate produced in the incubation, we employed a direct injection of the sample onto a preconcentration column. The obtained results indicated a high sensitivity of this method.     

DpgC is a metal- and cofactor-free 3,5-dihydroxyphenylacetyl-CoA 1,2-dioxygenase in the vancomycin biosynthetic pathway

3,5-Dihydroxyphenylglycine is a crucial amino acid monomer in the nonribosomal glycopeptide antibiotic vancomycin. This nonproteinogenic amino acid is constructed from malonyl-CoA by a set of four enzymes, DpgA-D, in the biosynthetic cluster. DpgC is an unusual metal-free, cofactor-free enzyme that consumes O(2) during the conversion of 3,5-dihydroxyphenylacetyl-CoA (DPA-CoA) to the penultimate intermediate 3,5-dihydroxyphenylglyoxylate (DPGx). We show that in anaerobic incubations, DpgC catalyzes the exchange of the C(2)-methylene hydrogens of DPA-CoA at unequal rates, consistent with enzyme-mediated formation of the substrate-derived C(2)-carbanion as an early intermediate. Incubations with (18)O(2) reveal that DpgC transfers both atoms of an O(2) molecule to DPGx product. This establishes DpgC as a 1,2-dioxygenase that mediates thioester cleavage by the oxygen transfer process. These results are consistent with a DPA-CoA C(2)-peroxy intermediate, followed by enzyme-directed alpha-peroxylactone formation and collapse by O-O bond cleavage.     

Aminoacyl-SNACs as small-molecule substrates for the condensation domains of nonribosomal peptide synthetases

BACKGROUND: Nonribosomal peptide synthetases (NRPSs) are large multidomain proteins that catalyze the formation of a wide range of biologically active natural products. These megasynthetases contain condensation (C) domains that catalyze peptide bond formation and chain elongation. The natural substrates for C domains are biosynthetic intermediates that are covalently tethered to thiolation (T) domains within the synthetase by thioester linkages. Characterizing C domain substrate specificity is important for the engineered biosynthesis of new compounds. RESULTS: We synthesized a series of aminoacyl-N-acetylcysteamine thioesters (aminoacyl-SNACs) and show that they are small-molecule substrates for NRPS C domains. Comparison of rates of peptide bond formation catalyzed by the C domain from enterobactin synthetase with various aminoacyl-SNACs as downstream (acceptor) substrates revealed high selectivity for the natural substrate analog L-Ser-SNAC. Comparing L- and D-Phe-SNACs as upstream (donor) substrates for the first C domain from tyrocidine synthetase revealed clear D- versus L-selectivity. CONCLUSIONS: Aminoacyl-SNACs are substrates for NRPS C domains and are useful for characterizing the substrate specificity of C domain-catalyzed peptide bond formation.     

Interrogation of global active site occupancy of a fungal iterative polyketide synthase reveals strategies for maintaining biosynthetic fidelity

Nonreducing iterative polyketide synthases (NR-PKSs) are responsible for assembling the core of fungal aromatic natural products with diverse biological properties. Despite recent advances in the field, many mechanistic details of polyketide assembly by these megasynthases remain unknown. To expand our understanding of substrate loading, polyketide elongation, cyclization, and product release, active site occupancy and product output were explored by Fourier transform mass spectrometry using the norsolorinic acid anthrone-producing polyketide synthase, PksA, from the aflatoxin biosynthetic pathway in Aspergillus parasiticus. Here we report the simultaneous observation of covalent intermediates from all catalytic domains of PksA from in vitro reconstitution reactions. The data provide snapshots of iterative catalysis and reveal an underappreciated editing function for the C-terminal thioesterase domain beyond its recently established synthetic role in Claisen/Dieckmann cyclization and product release. The specificity of thioesterase catalyzed hydrolysis was explored using biosynthetically relevant protein-bound and small molecule acyl substrates and demonstrated activity against hexanoyl and acetyl, but not malonyl. Processivity of polyketide extension was supported by the inability of a nonhydrolyzable malonyl analog to trap products of intermediate chain lengths and by the detection of only fully extended species observed covalently bound to, and as the predominant products released by, PksA. High occupancy of the malonyl transacylase domain and fast relative rate of malonyl transfer compared to starter unit transfer indicate that rapid loading of extension units onto the carrier domain facilitates efficient chain extension in a manner kinetically favorable to ultimate product formation.     

3D Structure Determination of an Unstable Transient Enzyme Intermediate by Paramagnetic NMR Spectroscopy

Enzyme catalysis relies on conformational plasticity, but structural information on transient intermediates is difficult to obtain. We show that the three‐dimensional (3D) structure of an unstable, low‐abundance enzymatic intermediate can be determined by nuclear magnetic resonance (NMR) spectroscopy. The approach is demonstrated for Staphylococcus aureus sortase A (SrtA), which is an established drug target and biotechnological reagent. SrtA is a transpeptidase that converts an amide bond of a substrate peptide into a thioester. By measuring pseudocontact shifts (PCSs) generated by a site‐specific cysteine‐reactive paramagnetic tag that does not react with the active‐site residue Cys184, a sufficient number of restraints were collected to determine the 3D structure of the unstable thioester intermediate of SrtA that is present only as a minor species under non‐equilibrium conditions. The 3D structure reveals structural changes that protect the thioester intermediate against hydrolysis.     

Utilization of alternate substrates by the first three modules of the epothilone synthetase assembly line

The epothilones, a family of macrolactone natural products produced by the myxobacterial species Sorangium cellulosum, are of current clinical interest as antitumor agents. Inspection of the structure of the epothilones suggests a hybrid polyketide/nonribosomal peptide biosynthetic origin, and the recent sequencing of the epothilone biosynthetic gene cluster has validated this proposal. Here we have examined unnatural substrates with the first two enzymes of the biosynthetic pathway, EpoA and EpoB, to investigate the enzymatic construction of alternate heterocyclic structures and the subsequent elongation of these products by the third enzyme of the pathway, EpoC. The epothilone biosynthetic machinery can utilize serine to install an oxazole in place of a thiazole in the epothilone structure and will tolerate functionalized donor groups from the EpoA-ACP domain to produce epothilone fragments modified at the C21 position. These studies with the early enzymes of the epothilone biosynthesis cluster suggest that combinatorial biosynthesis may be a viable means for producing a variety of epothilone analogues that incorporate diversity into the heterocycle starter unit.     

Biosynthesis of (-)-(1S,2R)-allocoronamic acyl thioester by an Fe(II)-dependent halogenase and a cyclopropane-forming flavoprotein

The biosynthetic gene cluster for the kutzneride family of hexapeptidolactones includes the four-gene cassette ktzABCD postulated to generate a nonproteinogenic amino acid. Encoded by this cassette are the nonheme FeII-dependent halogenase KtzD and the acyl-CoA dehydrogenase-like flavoprotein KtzA, proposed to work in conjunction with adenylating protein KtzB and carrier protein KtzC. In the present work, we report the in vitro reconstitution of this four-protein system and identify the final product as (1S,2R)-allocoronamic acid bound in thioester linkage to KtzC. Further analysis of KtzD and KtzA support a biosynthetic pathway that involves KtzD-mediated generation of a gamma-chloroisoleucyl intermediate which is cyclized to the final product by KtzA without redox participation of the bound flavin cofactor. This work introduces a new monomer for potential incorporation into nonribosomal peptides and validates the unique strategy for its biosynthesis.     

Genetically engineered synthesis and structural characterization of cobalt-precorrin 5A and -5B, two new intermediates on the anaerobic pathway to vitamin B12: definition of the roles of the CbiF and CbiG enzymes

Two new cobalt corrinoid intermediates, cobalt-precorrin 5A and cobalt-precorrin 5B, have been synthesized with the aid of overexpressed enzymes of the vitamin B(12) pathway of Salmonella entericaserovar typhimurium. These compounds were made in several regioselectively (13)C-labeled forms, and their structures have been established by multidimensional NMR spectroscopy. The addition of CbiF to the enzymes known to synthesize cobalt-precorrin 4 resulted in the formation of cobalt-precorrin 5A, and the inclusion of CbiG with CbiF produced cobalt-precorrin 5B, which has allowed us to define the role of these enzymes in the anaerobic biosynthetic pathway. CbiF is the C-11 methylase, and CbiG, an enzyme which shows homology with CobE of the aerobic pathway, is the gene product responsible for the opening of the ring A delta-lactone and extrusion of the "C(2)" unit. The discovery of these long-sought intermediates paves the way for defining the final stages of the anaerobic pathway. It is of considerable evolutionary interest that nature uses two distinct pathways to vitamin B(12), both conserved over several billion years and featuring completely different mechanisms for ring-contraction of the porphyrinoid to the corrinoid ring system. Thus the aerobic pathway utilizes molecular oxygen to trigger the events at C-20 leading to contraction and expulsion of the "C(2)" unit as acetic acid from a metal-free intermediate, whereas the anaerobic route features internal delivery of oxygen from a carboxylic acid terminus to C-20 followed by extrusion of the "C(2)" unit as acetaldehyde, using cobalt complexes as substrates.     

Chemoenzymatic synthesis of the polyketide macrolactone 10-deoxymethynolide

The polyketide synthase-derived pikromycin thioesterase (Pik TE) is unique in its ability to catalyze the cyclization of 12- and 14-membered macrolactones. In this investigation, the total synthesis of the natural hexaketide chain elongation intermediate as its N-acetyl cysteamine (NAC) thioester has been achieved, and its reaction with Pik TE demonstrated the ability of Pik TE to catalyze its macrolactonization to the natural product 10-deoxymethynolide. A steady-state kinetic analysis of the hexaketide chain intermediate with Pik TE was done. A preliminary substrate specificity study with unnatural hexaketide analogues was accomplished, demonstrating the importance of total synthesis in obtaining access to advanced polyketide intermediates. The results show the sensitivity of Pik TE to minor substrate modifications, and illustrate the potential use of thioesterases as versatile macrolactonization catalysts.     

The lyngbyatoxin biosynthetic assembly line: chain release by four-electron reduction of a dipeptidyl thioester to the corresponding alcohol

In comparison with the large number of nonribosomal peptide synthetases (NRPSs) that release their peptide products by hydrolytic cleavage of the peptide carrier protein (PCP) bound thioester, there are relatively few NRPSs that have been shown to use a nicotinamide cofactor to reduce this PCP-peptidyl thioester to an aldehyde or imine moiety. This work describes the first example of a reductase domain within a NRPS scaffold shown to reduce a PCP-peptidyl thioester to the corresponding primary alcohol, via an aldehyde intermediate, using two equivalents of reduced nicotinamide adenine dinucleotide phosphate (NADPH). By employing a ketone mimic of the aldehyde intermediate, as well as a specifically deuterated NADPH, it was further demonstrated that the pro-S hydride of the cofactor is transferred to the re face of the carbonyl group.     

Enzymatic desaturation of fatty acids: delta11 desaturase activity on cyclopropane acid probes

The formation of methylenecyclopropanes by enzymatic desaturation of 11-cyclopropylundecanoic acid (1) and its disubstituted derivatives cis- and trans-3-5 has been investigated using the Delta(11) desaturase of Spodoptera littoralis as model enzyme. Gas chromatography coupled to mass spectrometry analyses of methanolyzed lipidic extracts from tissues incubated with each probe revealed that all the cyclopropyl fatty acids were transformed into the corresponding 11-cyclopropylidene acids, except for compound trans-5 (5b), which was not desaturated at C11. The formation of methylenecyclopropane 9 as the only reaction product from 1 indicates that a potential radical intermediate is too short-lived to allow rearrangement reactions. Information on the Delta(11) desaturase substrate binding domain is provided considering the cyclopropyl probes 3-5 as conformationally restricted analogues of the straight-chain substrates.     

A theoretical study on the trans-addition intramolecular hydroacylation of 4-alkynals catalyzed by cationic rhodium complexes

The mechanism of the intramolecular hydroacylation reaction of 4-alkynals is studied for a 4-pentynal-[Rh(PH2CH2CH2PH2)]+ model system using MP2 calculations. The endo-cyclization to form a rhodacyclohexenone intermediate is kinetically less favorable than the exo-cyclization to form a rhodacyclopentanone intermediate. The kinetic preference toward the endo-cyclization is found to be enhanced by complexation of donor ligands (H2CO, NCH, and HCCH). The formation of cyclopentenone product proceeds via reductive elimination from one of the two rhodacyclohexenone intermediates, whereas the formation of cyclobutanone product from the two rhodacyclopentanone intermediates requires high activation energy. Addition of an acetylene stabilizes the highly electron-poor rhodacyclopentanone intermediate generated from exo-cyclization and leads to an insertion to give [4 + 2] annulation product, cyclohexenone. The role of a coordinating acetone solvent in the formation of cyclopentenone product is also discussed.     

Enzyme–Substrate Complementarity Governs Access to a Cationic Reaction Manifold in the P450BM3‐Catalysed Oxidation of Cyclopropyl Fatty Acids

The products of cytochrome P450_BM3‐catalysed oxidation of cyclopropyl‐containing dodecanoic acids are consistent with the presence of a cationic reaction intermediate, which results in efficient dehydrogenation of the rearranged probes by the enzyme. These results highlight the importance of enzyme–substrate complementarity, with a cationic intermediate occurring only when the probes used begin to diverge from ideal substrates for this enzyme. This also aids in reconciling literature reports supporting the presence of cationic intermediates with certain cytochrome P450 enzyme/substrate pairs.     

Substrate modulation of the properties and reactivity of the oxy-ferrous and hydroperoxo-ferric intermediates of cytochrome P450cam as shown by cryoreduction-EPR/ENDOR spectroscopy

EPR/ENDOR studies have been carried out on oxyferrous cytochrome P450cam one-electron cryoreduced by gamma-irradiation at 77 K in the absence of substrate and in the presence of a variety of substrates including its native hydroxylation substrate, camphor (a), and the alternate substrates, 5-methylenyl-camphor (b), 5,5-difluorocamphor (c), norcamphor (d), and adamantanone (e); the equivalent experiments have been performed on the T252A mutant complexed with a and b. The present study shows that the properties and reactivity of the oxyheme and of both the primary and the annealed intermediates are modulated by a bound substrate. This includes alterations in the properties of the heme center itself (g tensor; (14)N, (1)H, hyperfine couplings). It also includes dramatic changes in reactivity: the presence of any substrate increases the lifetime of hydroperoxoferri-P450cam (2) no less than ca. 20-fold. Among the substrates, b stands out as having an exceptionally strong influence on the properties and reactivity of the P450cam intermediates, especially in the T252A mutant. The intermediate, 2(T252A)-b, does not lose H(2)O(2), as occurs with 2(T252A)-a, but decays with formation of the epoxide of b. Thus, these observations show that substrate can modulate the properties of both the monoxygenase active-oxygen intermediates and the proton-delivery network that encompasses them.     

Macrodiolide Formation by the Thioesterase of a Modular Polyketide Synthase

Elaiophylin is an unusual C₂‐symmetric antibiotic macrodiolide produced on a bacterial modular polyketide synthase assembly line. To probe the mechanism and selectivity of diolide formation, we sought to reconstitute ring formation in vitro by using a non‐natural substrate. Incubation of recombinant elaiophylin thioesterase/cyclase with a synthetic pentaketide analogue of the presumed monomeric polyketide precursor of elaiophylin, specifically its N‐acetylcysteamine thioester, produced a novel 16‐membered C₂‐symmetric macrodiolide. A linear dimeric thioester is an intermediate in ring formation, which indicates iterative use of the thioesterase active site in ligation and subsequent cyclization. Furthermore, the elaiophylin thioesterase acts on a mixture of pentaketide and tetraketide thioesters to give both the symmetric decaketide diolide and the novel asymmetric hybrid nonaketide diolide. Such thioesterases have potential as tools for the in vitro construction of novel diolides.     

A synthetic model of the putative Fe(II)-iminobenzosemiquinonate intermediate in the catalytic cycle of o-aminophenol dioxygenases

The oxidative ring cleavage of aromatic substrates by nonheme Fe dioxygenases is thought to involve formation of a ferrous-(substrate radical) intermediate. Here we describe the synthesis of the trigonal-bipyramdial complex Fe((Ph2)Tp)(ISQ(tBu)) (2), the first synthetic example of an iron(II) center bound to an iminobenzosemiquinonate (ISQ) radical. The unique electronic structure of this S = 3/2 complex and its one-electron oxidized derivative ([3](+)) have been established on the basis of crystallographic, spectroscopic, and computational analyses. These findings further demonstrate the viability of Fe(2+)-ISQ intermediates in the catalytic cycles of o-aminophenol dioxygenases.     

Electron capture and transfer dissociation: Peptide structure analysis at different ion internal energy levels

We decoupled electron-transfer dissociation (ETD) and collision-induced dissociation of charge-reduced species (CRCID) events to probe the lifetimes of intermediate radical species in ETD-based ion trap tandem mass spectrometry of peptides. Short-lived intermediates formed upon electron transfer require less energy for product ion formation and appear in regular ETD mass spectra, whereas long-lived intermediates require additional vibrational energy and yield product ions as a function of CRCID amplitude. The observed dependencies complement the results obtained by double-resonance electron-capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ECD in a cryogenic ICR trap. Compared with ECD FT-ICR MS, ion trap MS offers lower precursor ion internal energy conditions, leading to more abundant charge-reduced radical intermediates and larger variation of product ion abundance as a function of vibrational post-activation amplitude. In many cases decoupled CRCID after ETD exhibits abundant radical c-type and even-electron z-type ions, in striking contrast to predominantly even-electron c-type and radical z-type ions in ECD FT-ICR MS and especially activated ion-ECD, thus providing a new insight into the fundamentals of ECD/ETD.