RESUMO
The H-cluster of [FeFe]-hydrogenase consists of a [4Fe-4S]H-subcluster linked by a cysteinyl bridge to a unique organometallic [2Fe]H-subcluster assigned as the site of interconversion between protons and molecular hydrogen. This [2Fe]H-subcluster is assembled by a set of Fe-S maturase enzymes HydG, HydE and HydF. Here we show that the HydG product [FeII(Cys)(CO)2(CN)] synthon is the substrate of the radical SAM enzyme HydE, with the generated 5'-deoxyadenosyl radical attacking the cysteine S to form a C5'-S bond concomitant with reduction of the central low-spin Fe(II) to the Fe(I) oxidation state. This leads to the cleavage of the cysteine C3-S bond, producing a mononuclear [FeI(CO)2(CN)S] species that serves as the precursor to the dinuclear Fe(I)Fe(I) center of the [2Fe]H-subcluster. This work unveils the role played by HydE in the enzymatic assembly of the H-cluster and expands the scope of radical SAM enzyme chemistry.
Assuntos
Hidrogenase/metabolismo , Compostos de Ferro/metabolismo , S-Adenosilmetionina/metabolismo , Thermotoga maritima/enzimologia , Biocatálise , Hidrogenase/química , Compostos de Ferro/química , Conformação Molecular , S-Adenosilmetionina/químicaRESUMO
S-Adenosyl methionine (SAM) is employed as a [4Fe-4S]-bound cofactor in the superfamily of radical SAM (rSAM) enzymes, in which one-electron reduction of the [4Fe-4S]-SAM moiety leads to homolytic cleavage of the S-adenosyl methionine to generate the 5'-deoxyadenosyl radical (5'dAdoâ¢), a potent H-atom abstractor. HydG, a member of this rSAM family, uses the 5'dAdo⢠radical to lyse its substrate, tyrosine, producing CO and CN that bind to a unique Fe site of a second HydG Fe-S cluster, ultimately producing a mononuclear organometallic Fe-l-cysteine-(CO)2CN complex as an intermediate in the bioassembly of the catalytic H-cluster of [Fe-Fe] hydrogenase. Here we report the use of non-native tyrosine substrate analogues to further probe the initial radical chemistry of HydG. One such non-native substrate is 4-hydroxy phenyl propanoic acid (HPPA) which lacks the amino group of tyrosine, replacing the CαH-NH2 with a CH2 at the C2 position. Electron paramagnetic resonance (EPR) studies show the generation of a strong and relatively stable radical in the HydG reaction with natural abundance and 13C2-HPPA, with appreciable spin density localized at C2. These results led us to try parallel experiments with the more oxidized non-native substrate coumaric acid, which has a C2=C3 alkene substitution relative to HPPA's single bond. Interestingly, the HydG reaction with the cis-p-coumaric acid isomer led to the trapping of a new radical EPR signal, and EPR studies using cis-p-coumaric acid along with isotopically labeled SAM reveal that we have for the first time trapped and characterized the 5'dAdo⢠radical in an actual rSAM enzyme reaction, here by using this specific non-native substrate cis-p-coumaric acid. Density functional theory energetics calculations show that the cis-p-coumaric acid has approximately the same C-H bond dissociation free energy as 5'dAdoâ¢, providing a possible explanation for our ability to trap an appreciable fraction of 5'dAdo⢠in this specific rSAM reaction. The radical's EPR line shape and its changes with SAM isotopic substitution are nearly identical to those of a 5'dAdo⢠radical recently generated by cryophotolysis of a prereduced [4Fe-4S]-SAM center in another rSAM enzyme, pyruvate formate-lyase activating enzyme, further supporting our assignment that we have indeed trapped and characterized the 5'dAdo⢠radical in a radical SAM enzymatic reaction by appropriate tuning of the relative radical free energies via the judicious selection of a non-native substrate.
RESUMO
The recently discovered futalosine-dependent menaquinone biosynthesis pathway employs radical chemistry for the naphthoquinol core assembly. Mechanistic studies on this pathway have resulted in the discovery of novel reaction motifs. MqnA is the first example of a chorismate dehydratase. MqnE is the first example of a radical SAM enzyme that catalyzes the addition of the 5'-deoxyadenosyl radical to the substrate double bond rather than hydrogen atom abstraction. Both MqnE and MqnC reaction sequences involve radical additions to a benzene ring followed by formation of an aryl radical anion intermediate. The enzymology of the tailoring reactions after dihydroxynaphthoic acid formation remains to be elucidated. Since the futalosine-dependent menaquinone biosynthesis pathway is absent in humans, mechanistic studies on this pathway may promote the development of new antibiotics.
Assuntos
Hidrolases/metabolismo , Nucleosídeos/metabolismo , Vitamina K 2/metabolismo , Ácido Corísmico/metabolismo , Humanos , Streptomyces coelicolor/enzimologia , Streptomyces coelicolor/metabolismoRESUMO
Aminofutalosine synthase (MqnE) is a radical SAM enzyme involved in the futalosine-dependent menaquinone biosynthetic pathway. Its ability to add the 5'-deoxyadenosyl radical to the substrate-rather than abstract a hydrogen atom-and to catalyze radical addition to a stable benzene ring gives it a unique place in the radical SAM superfamily and required the development of new strategies for trapping radical intermediates. This chapter describes the methodologies used for enzyme overexpression, purification, and in vitro reconstitution. We also describe the development of fast, radical triggered, carbon-halogen bond fragmentation reactions for the trapping of intermediates. We anticipate that these methods will be of general use in the study of other transient enzymatic radicals.
Assuntos
Alquil e Aril Transferases/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/metabolismo , Ensaios Enzimáticos/métodos , Nucleosídeos/metabolismo , Alquil e Aril Transferases/química , Alquil e Aril Transferases/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Biocatálise , Vias Biossintéticas , Clonagem Molecular/métodos , Radicais Livres/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/metabolismo , Thermus thermophilus/metabolismo , Vitamina K 2/metabolismoRESUMO
Aminofutalosine synthase (MqnE) is a radical SAM enzyme involved in the menaquinone biosynthetic pathway. In this communication, we propose a novel mechanism for this reaction involving the addition of the adenosyl radical to the substrate double bond to form a captodative radical followed by rearrangement and decarboxylation to form an aryl radical anion which is then oxidized by the [4Fe-4S]+2 cluster. Consistent with this proposal, we describe the trapping of the captodative radical and the aryl radical anion using radical triggered C-Br fragmentation reactions. We also describe the trapping of the captodative radical by replacing the vinylic carboxylic acid with an amide.