X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification - PubMed (original) (raw)

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

Peter J Goldman et al. Proc Natl Acad Sci U S A. 2013.

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 peptidyl-substrates, 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 cysteine-rich 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.

Keywords: iron–sulfur cluster fold; radical SAM dehydrogenase.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

anSME reaction. (1) Electron donation to the AdoMet radical cluster initiates homolysis of AdoMet and 5′dA• formation in the presence of bound substrate, (2) substrate radical generation, (3) deprotonation of the substrate Cys sidechain, (4) substrate oxidation, and (5) hydrolysis of the thioaldehyde intermediate yields the FGly moiety of the activated sulfatase.

Fig. 2.

Fig. 2.

Structure of anSMEcpe. (A) The AdoMet domain (magenta) contains the AdoMet cluster and the (β/α)6 partial TIM barrel. The SPASM domain (green) comprises most of the C-terminal segment and houses the remaining two [4Fe-4S] clusters. Two helices, α6a and α6′, are not part of either domain and are colored light blue. (B) Positions and distances between the three [4Fe-4S] clusters (stick representation with Fe in orange and S in yellow). (C) Topology of anSMEcpe. A, AdoMet cluster; I, Aux I; II, Aux II. (D) The SPASM domain, colored by the level of sequence homology between anSMEcpe and the other 280 members of TIGR04085. Conservation scores were calculated by the ConSurf server (40). Iron ligating cysteines are shown as spheres and labeled by their designation in C. Aux I and II are shown in stick representation and labeled.

Fig. 3.

Fig. 3.

Substrate peptide binding. (A) Cp18Cys (black) and Kp18Cys (gray) enter and exit the active site of anSMEcpe via the underside of the barrel. The Cβ carbon is 8.6 and 8.9 Å from Aux I and the AdoMet cluster, respectively. AdoMet and auxiliary clusters are shown in sticks with carbons in gray, oxygens in red, nitrogens in blue, sulfurs in yellow, and irons in orange. anSMEcpe β strands and SPASM domain are shown in ribbons and colored as in Fig. 2. (B) Substrate peptides bound to anSMEcpe and (C) bound to FGE (PDB ID code 2AIJ, blue) (7) were overlaid by the five residues encompassing the conserved sulfatase motif in each system and are shown in the same orientation.

Fig. 4.

Fig. 4.

anSME active site. (A) The active site of anSMEcpe. Sticks are displayed for AdoMet, target cysteine, and residues within 5 Å of the substrate cysteine Sγ. Distances as follows: AdoMet 5′C–cysteine Cβ, 4.1 Å; Y24–Sγ, 4.7 Å; D277–Sγ, 4.6 Å; Q64 – Sγ, 3.3 Å. Colored as in Fig. 3. (B) FGly and 5′dA production for the Y24F and D277N mutants. Displayed product formation is per μmol enzyme. *Wild-type data from ref. .

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