Mechanistic Study on the Reaction of a Radical SAM Dehydrogenase BtrN by Electron Paramagnetic Resonance Spectroscopy

Yokoyama, Kenichi; Ohmori, Daijiro; Kudo, Fumitaka; Eguchi, Tadashi

doi:10.1021/bi800509x

Cited by 48 publications

(61 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In BtrN, E189 forms a hydrogen bond/salt bridge with R152 ( Fig. 2A) After or concerted with deprotonation, collapse of the •C3-Oradical to the C3=O product necessitates loss of an electron, most likely to one of BtrN's [4Fe-4S] clusters (5,17,18,34). The AdoMet and Aux clusters are 8.6 and 9.6 Å away, respectively, from the C3 position of DOIA, making them both within range for a suitable electron transfer partner (35) (Fig.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry

Goldman

Grove²,

Booker

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The 2-deoxy-scyllo-inosamine (DOIA) dehydrogenases are key enzymes in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics. In contrast to most DOIA dehydrogenases, which are NAD-dependent, the DOIA dehydrogenase from Bacillus circulans (BtrN) is an S-adenosyl-L-methionine (AdoMet) radical enzyme. To examine how BtrN employs AdoMet radical chemistry, we have determined its structure with AdoMet and substrate to 1.56 Å resolution. We find a previously undescribed modification to the core AdoMet radical fold: instead of the canonical (β/α) 6 architecture, BtrN displays a (β 5 /α 4 ) motif. We further find that an auxiliary [4Fe-4S] cluster in BtrN, thought to bind substrate, is instead implicated in substrate-radical oxidation. High structural homology in the auxiliary cluster binding region between BtrN, fellow AdoMet radical dehydrogenase anSME, and molybdenum cofactor biosynthetic enzyme MoaA provides support for the establishment of an AdoMet radical structural motif that is likely common to ∼6,400 uncharacterized AdoMet radical enzymes.radical SAM enzyme | iron-sulfur cluster fold | twitch domain D ue to mounting antibiotic resistance, the discovery and/or modification of antibiotic compounds to fight bacterial infection is a vital task of our time (1). Understanding antibiotic biosynthesis is an important first step in being able to engineer a new wave of therapeutic molecules. Aminoglycosides are a class of antibiotics that inhibit protein synthesis by interacting with the 30S subunit of the bacterial ribosome and have had considerable success in the clinical setting (2). Most FDA-approved aminoglycosides, including neomycin, gentamicin, and kanamycin, share a common glucose-6-phosphate-derived 2-deoxystreptamine (DOS) structural core (3) (blue in Scheme 1). Although the majority of aminoglycoside-producing bacteria use similar reaction sequences to biosynthesize this DOS core, including the penultimate two-electron oxidation of 2-deoxy-scyllo-inosamine (DOIA) to amino-dideoxy-scyllo-inosose (amino-DOI) (Scheme 1, A), identification of the enzymes responsible has not always been straightforward. For example, the btrE gene product in the butirosin B producing Bacillus circulans was proposed to be an NADdependent enzyme responsible for the generation of amino-DOI, as in other aminoglycoside pathways (4). This hypothesis proved incorrect upon further sequence and biochemical analysis (5, 6). Instead, Yokoyama et al. found that production of amino-DOI required another enzyme, BtrN, an S-adenosyl-L-methionine (AdoMet, SAM) radical dehydrogenase (5). Here, we report structures of catalytic and noncatalytic forms of BtrN, providing a structural basis for the unique reaction it performs.The AdoMet radical enzyme family catalyzes a diverse array of radical-based reactions, including sulfur insertions, complex chemical transformations and rearrangements, DNA and RNA modifications, and, in the case of BtrN, dehydrogenation (7). BtrN is one of two known members of the dehydrogenase subfamily of ...

show abstract

Section: Discussionmentioning

confidence: 99%

“…In BtrN, hydrogen atom abstraction by 5′dA• results in a substrate-based radical intermediate localized at position C3 (5,17). Subsequent deprotonation and oxidation of this intermediate yields the amino-DOI product (Scheme 1, A).…”

mentioning

confidence: 99%

X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry

Goldman

Grove²,

Booker

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Indeed, substrate radical intermediates have also been trapped and characterized by EPR in the cases of BtrN, 25 ethanolamine ammonia lyase (EAL) 26 and diol dehydratase 27 . Like DesII, BtrN is also a radical SAM dehydrogenase involving a cyclitol substrate ( 10 ) with a β -hydroxy functionality adjacent to the carbon that is oxidized.…”

Section: The Substrate Radicalmentioning

confidence: 99%

Mechanistic Enzymology of the Radical SAM Enzyme DesII

Ruszczycky

Liu

2015

Israel Journal of Chemistry

View full text Add to dashboard Cite

DesII is a member of the radical SAM family of enzymes that catalyzes radical-mediated transformations of TDP-4-amino-4,6-didexoy-D-glucose as well as other sugar nucleotide diphosphates. Like nearly all radical SAM enzymes, the reactions begin with the reductive homolysis of SAM to produce a 5′-deoxyadenosyl radical which is followed by regiospecific hydrogen atom abstraction from the substrate. What happens next, however, depends on the nature of the substrate radical so produced. In the case of the biosynthetically relevant substrate, a radical-mediated deamination ensues; however, when this amino group is replaced with a hydroxyl, one instead observes dehydrogenation. The factors that govern the fate of the initially generated substrate radical as well as the mechanistic details underlying these transformations have been a key focus of research into the chemistry of DesII. This review will discuss recent discoveries pertaining to the enzymology of DesII, how it may relate to understanding other radical-mediated lyases and dehydrogenases and the working hypotheses currently being investigated regarding the mechanism of DesII catalysis.

show abstract

“…The AdoMet radical dehydrogenase subfamily includes anSMEs and the carbohydrate natural product biosynthetic enzyme BtrN (18,19). Interestingly, both enzymes harbor additional [4Fe-4S] clusters that are necessary for turnover (20).…”

mentioning

confidence: 99%

X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification

Goldman¹,

Grove²,

Sites³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

109

197

View full text Add to dashboard Cite

Arylsulfatases require a maturating enzyme to perform a co-or posttranslational modification to form a catalytically essential formylglycine (FGly) residue. In organisms that live aerobically, molecular oxygen is used enzymatically to oxidize cysteine to FGly. Under anaerobic conditions, S-adenosylmethionine (AdoMet) radical chemistry is used. Here we present the structures of an anaerobic sulfatase maturating enzyme (anSME), both with and without peptidyl-substrates, at 1.6-1.8 Å resolution. We find that anSMEs differ from their aerobic counterparts in using backbone-based hydrogen-bonding patterns to interact with their peptidylsubstrates, leading to decreased sequence specificity. These anSME structures from Clostridium perfringens are also the first of an AdoMet radical enzyme that performs dehydrogenase chemistry. Together with accompanying mutagenesis data, a mechanistic proposal is put forth for how AdoMet radical chemistry is coopted to perform a dehydrogenation reaction. In the oxidation of cysteine or serine to FGly by anSME, we identify D277 and an auxiliary [4Fe-4S] cluster as the likely acceptor of the final proton and electron, respectively. D277 and both auxiliary clusters are housed in a cysteinerich C-terminal domain, termed SPASM domain, that contains homology to ∼1,400 other unique AdoMet radical enzymes proposed to use [4Fe-4S] clusters to ligate peptidyl-substrates for subsequent modification. In contrast to this proposal, we find that neither auxiliary cluster in anSME bind substrate, and both are fully ligated by cysteine residues. Instead, our structural data suggest that the placement of these auxiliary clusters creates a conduit for electrons to travel from the buried substrate to the protein surface.iron-sulfur cluster fold | radical SAM dehydrogenase

show abstract

Mechanistic Study on the Reaction of a Radical SAM Dehydrogenase BtrN by Electron Paramagnetic Resonance Spectroscopy

Cited by 48 publications

References 55 publications

X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry

X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry

Mechanistic Enzymology of the Radical SAM Enzyme DesII

X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification

Contact Info

Product

Resources

About