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

Abstract

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

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

“…S2 and S3). In addition, the fold contains a basic residue, H117, that interacts with the carboxyl group of AdoMet, as seen in HemN (29), HydE (30), PylB (31), and anSMEcpe (20), and another hydrogen bond common to nearly all AdoMet radical members, between the N6 position of AdoMet and the carbonyl of Y22, the hydrophobic residue of the AdoMet radical cluster binding motif (16) (Fig. S3).…”

“…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.…”

“…Direct ligation of substrate to an Aux cluster became an attractive hypothesis for the role of these clusters, as it would aid in both deprotonation and oxidation of the substrate intermediate (9,18). However, the recently determined structure of anSME from Clostridium perfringens (anSMEcpe) solved in complex with a peptide substrate shows that these Aux clusters are fully ligated by cysteine residues from the protein and do not play a direct role in substrate binding (20).…”

“…Due to its partial-SPASM makeup, this substructure was named a "twitch" domain (20). Given that BtrN contains these sequence elements (CX 9-15 GX 4 CX n ), it was hypothesized to contain its Aux cluster in a twitch domain similar to MoaA (20). Interestingly, the Aux cluster in MoaA has an available iron ligation site that, unlike in anSMEcpe, is used to bind substrate (27,28).…”

“…These conserved features include a β hairpin surrounded by iron ligating cysteine positions and followed by a helical region. Due to its partial-SPASM makeup, this substructure was named a "twitch" domain (20). Given that BtrN contains these sequence elements (CX 9-15 GX 4 CX n ), it was hypothesized to contain its Aux cluster in a twitch domain similar to MoaA (20).…”