Crystal structure of the
            <i>S</i>
            -adenosylmethionine-dependent enzyme MoaA and its implications for molybdenum cofactor deficiency in humans

179

280

Pyruvate formate-lyase activating enzyme generates a stable and catalytically essential glycyl radical on G 734 of pyruvate formatelyase via the direct, stereospecific abstraction of a hydrogen atom from pyruvate formate-lyase. The activase performs this remarkable feat by using an iron-sulfur cluster and S-adenosylmethionine (AdoMet), thus placing it among the AdoMet radical superfamily of enzymes. We report here structures of the substrate-free and substrate-bound forms of pyruvate formate-lyase-activating enzyme, the first structures of an AdoMet radical activase. To obtain the substrate-bound structure, we have used a peptide substrate, the 7-mer RVSGYAV, which contains the sequence surrounding G 734 . Our structures provide fundamental insights into the interactions between the activase and the G 734 loop of pyruvate formate-lyase and provide a structural basis for direct and stereospecific H atom abstraction from the buried G 734 of pyruvate formate-lyase.crystallography ͉ metalloprotein ͉ radical chemistry ͉ S-adenosylmethionine ͉ iron-sulfur cluster

supporting

confidence: 80%

Structural basis for glycyl radical formation by pyruvate formate-lyase activating enzyme

Vey

Yang²,

Li³

et al. 2008

179

280

“…While the AdoMet domain of anSMEcpe is very similar to other members of the AdoMet radical family, the C-terminal SPASM domain is structurally similar (rmsd 6.3 Å) to only one other AdoMet radical protein, MoaA (33). Like anSMEcpe, MoaA ligates a C-terminal auxiliary cluster that overlays well with anSMEcpe's Aux I (Fig.…”

Section: Resultsmentioning

confidence: 85%

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

Goldman¹,

Grove²,

Sites³

et al. 2013

112

197

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

“…3). In addition to BtrN, MoaA contains a twitch domain with the same C-terminal secondary structural elements as BtrN and anSMEcpe (25) (Fig. 3 and Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The name SPASM derives from the biochemically characterized members of this subfamily, AlbA (22), PqqE (23), anSMEs (9,19), and MtfC (24), involved in subtilosin A, pyrroloquinoline quinone, anaerobic sulfatase, and mycofactocin maturation, respectively. Surprisingly, the anSME structure revealed similarities between the SPASM domain (20) and the Aux cluster-binding domain of molybdopterin biosynthetic enzyme MoaA, an AdoMet radical enzyme with a nonpeptide substrate (25,26). These conserved features include a β hairpin surrounded by iron ligating cysteine positions and followed by a helical region.…”

mentioning

confidence: 99%

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

Goldman

Grove²,

Booker

et al. 2013

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 ...