Guidelines for tunneling in enzymes

Moser, Christopher C.; Anderson, J. L. Ross; Dutton, P. Leslie

doi:10.1016/j.bbabio.2010.04.441

Cited by 137 publications

(178 citation statements)

References 106 publications

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“…After hydrogen abstraction and thiol deprotonation, formation of the thioaldehyde drives oxidation of the Cβ, requiring an electron acceptor to complete catalysis. Cβ lies 8.6 and 8.9 Å from Aux I and the AdoMet cluster, respectively, indicating both clusters are within suitable distance to be electron transfer partners (35). If an electron transfer event results in the reduction of Aux I, removing the electron from the system would likely require transfer from Aux I to Aux II, as peptide binding provides a barrier between Aux I and solvent (Fig.…”

Section: Resultsmentioning

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.

112

197

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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

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Section: Resultsmentioning

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.

112

197

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“…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. S5).…”

Section: Discussionmentioning

confidence: 99%

“…To reoxidize the Aux cluster in BtrN, electron transfer to the AdoMet cluster has been proposed, which would ready this cluster for the next round of catalysis (18). At 15.9 Å, however, the distance between the two clusters is on the longer side for facile electron transfer (35), suggesting that, like in anSMEcpe, electron transfer between the Aux and AdoMet clusters does not occur (20). We therefore propose that, following turnover in BtrN, a partner protein reoxidizes the reduced Aux cluster directly, without an internal electron transfer event.…”

Section: Discussionmentioning

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.

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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 ...

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“…These reactions occur via the formation of competent FNR-substrate complexes with NAD(P)H and Fd. For the redox reactions to occur with a rapid turnover rate, appropriate positioning of the cofactors and reacting atoms/groups for hydride and electron tunneling in the complexes, and tuning of the redox properties of the redox centers are required (Moser et al 2010). In this sense, comparative studies of the different types of FNRs should contribute to increasing our understanding of the structure-function relationship of FNRs.…”

Section: Introductionmentioning

confidence: 99%

Replacement of Tyr50 stacked on the si-face of the isoalloxazine ring of the flavin adenine dinucleotide prosthetic group modulates Bacillus subtilis ferredoxin-NADP+ oxidoreductase activity toward NADPH

et al. 2015

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Guidelines for tunneling in enzymes

Cited by 137 publications

References 106 publications

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

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

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

Replacement of Tyr50 stacked on the si-face of the isoalloxazine ring of the flavin adenine dinucleotide prosthetic group modulates Bacillus subtilis ferredoxin-NADP+ oxidoreductase activity toward NADPH

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