Radical SAM catalysis via an organometallic intermediate with an Fe–[5′-C]-deoxyadenosyl bond

Horitani, Masaki; Shisler, Krista A.; Broderick, William E.; Hutcheson, Rachel U.; Duschene, Kaitlin S.; Marts, Amy R.; Hoffman, Brian M.; Broderick, Joan B.

doi:10.1126/science.aaf5327

Cited by 120 publications

(202 citation statements)

References 39 publications

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“…Our studies here thus have revealed that the [4Fe-4S] cluster in a radical SAM enzyme can be tuned to cleave any one of the three bonds to the sulfonium sulfur of SAM or analogues. Together with recent studies on PFL-AE and Ph Dph2, 17,29 our study provides compelling evidence for the versatility of these [4Fe-4S] clusters.…”

supporting

confidence: 81%

“…This intermediate, could be compared to the catalytic organometallic intermediate in pyruvate formate lyase-activate enzyme (PFL-AE). 17 Both intermediates are formed by substrate (SAM or SAM analogue) cleavage with bonding of the nascent radical/cleavage product to the [4Fe-4S] cluster, with the difference being whether the Fe or the S atom in the cluster is directly bonded.…”

mentioning

confidence: 99%

“…17 Although recently the formation of low level of 5′-deoxy-5′-thioadenosine, a reaction product of 5′-deoxyadenosyl radical with one sulfur in the oxidized cluster, was reported in NosL study. The iron sulfur cluster was inactivated instead of playing a catalytic role.…”

mentioning

confidence: 99%

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Substrate-Dependent Cleavage Site Selection by Unconventional Radical S-Adenosylmethionine Enzymes in Diphthamide Biosynthesis

et al. 2017

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S -adenosylmethionine (SAM) has a sulfonium ion with three distinct C-S bonds. Conventional radical SAM enzymes use a [4Fe–4S] cluster to homolytically cleave the C5′,adenosine-S bond of SAM to generate a 5′-deoxyadenosyl radical, which catalyzes various downstream chemical reactions. Radical SAM enzymes involved in diphthamide biosynthesis, such as Pyrococcus horikoshii Dph2 (PhDph2) and yeast Dph1-Dph2 instead cleave the Cγ,Met–S bond of methionine to generate the 3-amino-3-carboxylpropyl radical. We here show that radical SAM enzymes can be tuned to cleave the third C-S bond to the sulfonium sulfur by changing the structure of SAM. With a decarboxyl SAM analogue (dc-SAM), PhDph2 cleaves the Cmethyl–S bond, forming 5′-deoxy-5′-(3-aminopropylthio) adenosine (dAPTA, 1). The methyl cleavage activity, like the cleavage of the other two C-S bonds, is dependent on the presence of a [4Fe-4S]+ cluster. Electron-nuclear double resonance (ENDOR) and mass spectroscopy data suggests that mechanistically, one of the S atoms in the [4Fe-4S] cluster captures the methyl group from dc-SAM, forming a distinct EPR-active intermediate, which can transfer the methyl group to nucleophiles such as dithiothreitol. This reveals that the [4Fe-4S] cluster in a radical SAM enzyme can be tuned to cleave any one of the three bonds to the sulfonium sulfur of SAM or analogues, and is the first demonstration that a radical SAM enzyme could switch from an Fe-based one electron transfer reaction to a S-based two electron transfer reaction in a substrate-dependent manner. This study thus provides a remarkable illustration of the versatile reactivity of Fe-S clusters.

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confidence: 81%

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confidence: 99%

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Substrate-Dependent Cleavage Site Selection by Unconventional Radical S-Adenosylmethionine Enzymes in Diphthamide Biosynthesis

et al. 2017

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“…11,19 However, similar organometallic complex had not been demonstrated for RS enzymes until very recently, when the [4Fe-4S] 2+ cluster of the RS enzyme, pyruvate formatelyase activating enzyme is shown to react with the 5′-dA• to form a highly reactive organometallic intermediate. 20 This study and our current result suggest that the unique iron of [4Fe-4S] clusters in RS enzymes can readily form a C-Fe complex. Such a property not only may help to explain the unusual chemistries catalyzed by the [4Fe-4S]-containing RS enzymes, but also may open up new avenues to tune the activity or develop new chemistry for this enzyme class or for other [4Fe-4S]-containing enzymes.…”

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confidence: 64%

Organometallic Complex Formed by an Unconventional Radical S-Adenosylmethionine Enzyme

et al. 2016

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Pyrococcus horikoshii Dph2 (PhDph2) is an unusual radical SAM enzyme involved in the first step of diphthamide biosynthesis. It catalyzes the reaction by cleaving S-adenosylmethionine (SAM) to generate a 3-amino-3-carboxy propyl (ACP) radical. To probe the reaction mechanism, we synthesized a SAM analog (SAMCA), in which the ACP group of SAM is replaced with a 3-carboxyallyl group. SAMCA is cleaved by PhDph2, yielding a paramagnetic (S = 1/2) species, which is assigned to a complex formed between the reaction product, α-sulfinyl-3-butenoic acid, and the [4Fe-4S] cluster. Electron-nuclear double resonance (ENDOR) measurements with 13C and 2H isotopically labeled SAMCA support a π-complex between the C=C double bond of α-sulfinyl-3-butenoic acid, and the unique iron of the [4Fe-4S] cluster. This is the first example of a radical SAM related [4Fe-4S]+ cluster forming an organometallic complex with an alkene, shedding additional light into the mechanism of PhDph2 and expanding our current notions for the reactivity of [4Fe-4S] clusters in radical SAM enzymes.

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“…Figure S1). 12 Liu and coworkers showed that the H4′ atom of 1 is abstracted by 5′-dAdo• to give 5′-deoxyadenosine (5′-dAdo) as one of products in addition to the dehydrated product (Figure 2). 6 The reaction of AprD4/AprD3 with 1, 2, kanamycin C (3) and kanamycin B (4) produced similar amounts of 5′-dAdo, whereas only a small amount of 5′-dAdo was detected with 2′-N-acetylparomamine (5) and paromomycin (6) (Figure 3 and Table 1).…”

Section: Enzyme Reaction In the Presence Of Deuterium Oxidementioning

confidence: 99%

Substrate specificity of radical S-adenosyl-l-methionine dehydratase AprD4 and its partner reductase AprD3 in the C3′-deoxygenation of aminoglycoside antibiotics

2016

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A radical S-adenosyl-L-methionine dehydratase AprD4 and an NADPH-dependent reductase AprD3 are responsible for the C3′-deoxygenation of pseudodisaccharide paromamine in the biosynthesis of apramycin. These enzymes are involved in the construction of the characteristic structural motif that is not modified by 3′-phosphotransferase in aminoglycoside-resistant bacterial strains. AprD4 catalyzes the C3′-dehydration of paromamine via a radical-mediated reaction mechanism to give 4′-oxolividamine, which is then reduced by AprD3 with NADPH to afford lividamine. In the present study, the substrate specificity of this unique combination of enzymes has been investigated. AprD4 was found to recognize paromamine, neamine, kanamycin C, and kanamycin B to afford 5′-deoxyadenosine as one of products during the C3′-dehydration of aminoglycosides, but not 2′-N-acetylparomamine and paromomycin. Only paromamine and kanamycin C were converted to the corresponding C3′-deoxygenated compounds by AprD4 and AprD3. AprD3 recognizes the 4′-oxolividamine moiety, including the pseudotrisaccharide kanamycin C, and seems to reject the amino group at C6′ of neamine and kanamycin B. Chirally deuterium-labeled NADPH was used to identify that that AprD3 transfers the pro-S hydrogen atom of NADPH when reducing 4′-oxolividamine to give lividamine.

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Radical SAM catalysis via an organometallic intermediate with an Fe–[5′-C]-deoxyadenosyl bond

Cited by 120 publications

References 39 publications

Substrate-Dependent Cleavage Site Selection by Unconventional Radical S-Adenosylmethionine Enzymes in Diphthamide Biosynthesis

Substrate-Dependent Cleavage Site Selection by Unconventional Radical S-Adenosylmethionine Enzymes in Diphthamide Biosynthesis

Organometallic Complex Formed by an Unconventional Radical S-Adenosylmethionine Enzyme

Substrate specificity of radical S-adenosyl-l-methionine dehydratase AprD4 and its partner reductase AprD3 in the C3′-deoxygenation of aminoglycoside antibiotics

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