Anaerobic Sulfatase-maturating Enzymes, First Dual Substrate Radical S-Adenosylmethionine Enzymes

Benjdia, Alhosna; Subramanian, Sowmya; Leprince, Jérôme; Vaudry, Hubert; Johnson, Michael K.; Berteau, Olivier

doi:10.1074/jbc.m710074200

Cited by 65 publications

(97 citation statements)

References 35 publications

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“…S9) suggests that this twitch/SPASM domain will be a common feature to ∼6,400 uncharacterized AdoMet radical enzymes, comprising nearly 16% of the superfamily (40). Although it is difficult to predict iron sulfur cluster content in these twitch/SPASM subfamily members, all currently characterized members contain at least one Aux [4Fe-4S] cluster (8,9,18,20,(22)(23)(24)41).…”

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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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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“…In contrast to the numerous sulfatases found in this bacterium, only one gene (BT0238) encoding an anSME was detected based on a search of all known sulfatase-maturating enzymes (anSME or formylglycine-generating enzyme) present in the B. thetaiotaomicron genome (BLASTP E-value Ͻ10 Ϫ10 ). We recently demonstrated that BT0238 encodes indeed an authentic anSME (14,16). In other organisms, such as Escherichia coli K12 and Vibrio species, an anSME is commonly present in an operon that also contains a co-expressed sulfatase.…”

Section: Identification Of Putative Sulfatases In Bacteroidesmentioning

confidence: 99%

Sulfatases and a Radical S-Adenosyl-l-methionine (AdoMet) Enzyme Are Key for Mucosal Foraging and Fitness of the Prominent Human Gut Symbiont, Bacteroides thetaiotaomicron

Benjdia

Martens

Gordon

et al. 2011

Journal of Biological Chemistry

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133

134

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The large-scale application of genomic and metagenomic sequencing technologies has yielded a number of insights about the metabolic potential of symbiotic human gut microbes. Nevertheless, the molecular basis of the interactions between commensal bacteria and their host remained to be investigated. Bacteria colonizing the mucosal layer that overlies the gut epithelium are exposed to highly sulfated glycans (i.e. mucin and glycosaminoglycans). These polymers can serve as potential nutrient sources, but their high sulfate content usually prevents their degradation. Commensal bacteria such as Bacteroides thetaiotaomicron possess more predicted sulfatase genes than in the human genome, the physiological functions of which are largely unknown. To be active, sulfatases must undergo a critical post-translational modification catalyzed in anaerobic bacteria by the radical AdoMet enzyme anaerobic sulfatase-maturating enzyme (anSME). In the present study, we have tested the role of this pathway in Bacteroides thetaiotaomicron which, in addition to 28 predicted sulfatases, possesses a single predicted anSME. In vitro studies revealed that deletion of the gene encoding its anSME (BT0238) results in loss of sulfatase activity and impaired ability to use sulfated polysaccharides as carbon sources. Co-colonization of formerly germ-free mice with both isogenic strains (i.e. wild-type or ⌬anSME), or invasion experiments involving introduction of one followed by the other strain established that anSME activity and the sulfatases activated via this pathway, are important fitness factors for B. thetaiotaomicron, especially when mice are fed a simple sugar diet that requires this saccharolytic bacterium to adaptively forage on host glycans as nutrients. Whole genome transcriptional profiling of wild-type and the anSME mutant in vivo revealed that loss of this enzyme alters expression of genes involved in mucin utilization and that this disrupted ability to access mucosal glycans likely underlies the observed pronounced colonization defect. Comparative genomic analysis reveals that 100% of 46 fully sequenced human gut Bacteroidetes contain homologs of BT0238 and genes encoding sulfatases, suggesting that this is an important and evolutionarily conserved feature for bacterial adaptation to life in this habitat.

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“…Efficient generation of the substrate radical (12→14) is expected to require the enzyme to be in the appropriate ionization state due to the nonunit value of D2O « V rad at suboptimal pH. Following formation of the substrate radical 14, it can subsequently partition forward to form the oxidized product (16), partition backward to regenerate the Michaelis complex (12), or undergo protonation to form a dead-end complex also observable by EPR. The large forward commitment on D2O ðV =KÞ Sub is consistent with slow partitioning of 14 back to the Michaelis complex (12) vs. oxidation of 14 to 16, but it may also be explained by sticky substrate binding.…”

Section: +1mentioning

confidence: 99%

“…In this mechanism, DesII possesses an active site base that can exist in either a protonated or unprotonated state in the free enzyme (11, or binary complex with SAM). On binding of SAM (1) and substrate (6), a hydrogen bond is formed between this active site residue and the C3 hydroxyl group of TDP-D-quinovose in the ternary Michaelis complex (12). Disruption of this as well as any other pH-sensitive interactions in the protonated form of the enzyme may then lead to a weaker Michaelis complex and may help to explain the smaller value observed for p K V =K app vs. p K V app .…”

Section: +1mentioning

confidence: 99%

EPR-kinetic isotope effect study of the mechanism of radical-mediated dehydrogenation of an alcohol by the radical SAM enzyme DesII

Ruszczycky

Choi

Liu

2013

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

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The radical S-adenosyl-L-methionine enzyme DesII from Streptomyces venezuelae is able to oxidize the C3 hydroxyl group of TDP-D-quinovose to the corresponding ketone via an α-hydroxyalkyl radical intermediate. It is unknown whether electron transfer from the radical intermediate precedes or follows its deprotonation, and answering this question would offer considerable insight into the mechanism by which the small but important class of radical-mediated alcohol dehydrogenases operate. This question can be addressed by measuring steady-state kinetic isotope effects (KIEs); however, their interpretation is obfuscated by the degree to which the steps of interest limit catalysis. To circumvent this problem, we measured the solvent deuterium KIE on the saturating steady-state concentration of the radical intermediate using electron paramagnetic resonance spectroscopy. The resulting value, 0:22 ± 0:03, when combined with the solvent deuterium KIE on the maximum rate of turnover (V) of 1:8 ± 0:2, yielded a KIE of 8 ± 2 on the net rate constant specifically associated with the α-hydroxyalkyl radical intermediate. This result implies that electron transfer from the radical intermediate does not precede deprotonation. Further analysis of these isotope effects, along with the pH dependence of the steady-state kinetic parameters, likewise suggests that DesII must be in the correct protonation state for initial generation of the α-hydroxyalkyl radical. In addition to providing unique mechanistic insights, this work introduces a unique approach to investigating enzymatic reactions using KIEs.T he enzyme-catalyzed dehydrogenation of an alcohol to a carbonyl is one of the most common and fundamental reactions of both primary metabolism and secondary metabolism. In the majority of cases studied, these reactions proceed via hydride transfer to an organic cofactor, such as the nicotinamide moiety of NAD(P) + or the isoalloxazine group of FAD among others (1, 2). In contrast, much less is understood regarding radical-mediated dehydrogenation of an alcohol, because only a handful of enzyme examples have been described. Of the radical alcohol dehydrogenases, galactose oxidase is the most extensively studied, and it is known to use a Cu II ion coordinated to a proteinderived 3′-(S-cysteinyl)tyrosyl radical to catalyze dehydrogenation of the C6 hydroxyl group of galactose (3, 4). In contrast, BtrN and the anaerobic sulfatase maturating enzymes (anSMEs) are radical S-adenosyl-L-methionine (SAM) enzymes (5-7) responsible for the dehydrogenation of the C3 hydroxyl group of 2-deoxy-scylloinosamine (8, 9) and the hydroxyl/sulfhydryl moieties of serine/ cysteine residues (10-12), respectively. In addition to the primary [4Fe-4S] 1+ cluster, which reduces SAM (1) to methionine (2) and the 5′-deoxyadenosyl radical (17; see Fig. 4) to initiate radical catalysis, BtrN (13,14) and anSMEs (15-18) possess one or more auxiliary [4Fe-4S] aux clusters, which have been proposed to coordinate the hydroxyl group to be oxidized in the substrate.The enzyme...

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Anaerobic Sulfatase-maturating Enzymes, First Dual Substrate Radical S-Adenosylmethionine Enzymes

Cited by 65 publications

References 35 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

Sulfatases and a Radical S-Adenosyl-l-methionine (AdoMet) Enzyme Are Key for Mucosal Foraging and Fitness of the Prominent Human Gut Symbiont, Bacteroides thetaiotaomicron

EPR-kinetic isotope effect study of the mechanism of radical-mediated dehydrogenation of an alcohol by the radical SAM enzyme DesII

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