Letters pubs.acs.org/acschemicalbiology
Characterization of Choline Trimethylamine-Lyase Expands the Chemistry of Glycyl Radical Enzymes Smaranda Craciun, Jonathan A. Marks, and Emily P. Balskus* Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, United States S Supporting Information *
ABSTRACT: The recently identified glycyl radical enzyme (GRE) homologue choline trimethylamine-lyase (CutC) participates in the anaerobic conversion of choline to trimethylamine (TMA), a widely distributed microbial metabolic transformation that occurs in the human gut and is linked to disease. The proposed biochemical function of CutC, C−N bond cleavage, represents new reactivity for the GRE family. Here we describe the in vitro characterization of CutC and its activating protein CutD. We have observed CutD-mediated formation of a glycyl radical on CutC using EPR spectroscopy and have demonstrated that activated CutC processes choline to trimethylamine and acetaldehyde. Surveys of potential alternate CutC substrates uncovered a strict specificity for choline. Homology modeling and mutagenesis experiments revealed essential CutC active site residues. Overall, this work establishes that CutC is a GRE of unique function and a molecular marker for anaerobic choline metabolism.
N
associated GRE (CutC) and a GRE activating protein (CutD). We confirmed that CutC and CutD were responsible for the initial C−N bond fragmentation that generates TMA and acetaldehyde (Figure 1b) using genetics and heterologous expression.3 As this transformation impacts human disease and represents a new reaction for the GRE family, we decided to further examine the activity of CutC and CutD in vitro. Understanding the enzymatic chemistry involved in TMA generation, particularly the features that distinguish CutC from other GREs, will facilitate identification of this enzymatic activity in bacterial isolates and microbial communities, which will in turn help to decipher its role in habitats like the human gut. The GREs characterized to date are hypothesized to share certain mechanistic features (Figure 1c).2 The glycine-centered radical required for enzyme function is generated by the action of a separate activating protein, a member of the radical Sadenosylmethionine (SAM) enzyme family.11 These [4Fe−4S] cluster-containing enzymes homolytically cleave SAM, generating a 5′-deoxyadenosyl radical that abstracts an α-hydrogen atom from an active site glycine of a partner GRE. Substrate binding may trigger reaction of the glycyl radical with a nearby cysteine residue, possibly generating a thiyl radical that may initiate the reaction through formation of a substrate-based radical. Following further reaction or rearrangement, a productcentered radical may react with the active site cysteine, regenerating the thiyl radical. The catalytic glycine and cysteine
ature harnesses radical chemistry to accomplish many challenging biochemical transformations.1,2 Glycyl radical enzymes (GREs) utilize protein-based radical intermediates to catalyze a variety of reactions, including nucleotide reduction (class III ribonucleotide reductase (RNR)), C−C bond formation (benzylsuccinate synthase (BSS)), C−C bond cleavage (pyruvate formate-lyase (PFL) and 4-hydroxyphenylacetate decarboxylase (4-HPAD)), and dehydration (B12independent glycerol dehydratase (GDH)).2 Here we report the in vitro characterization of a new GRE from human gut bacteria, the C−N bond-cleaving enzyme choline trimethylamine (TMA)-lyase (CutC).3 This work reveals the exquisite selectivity of CutC for choline cleavage and provides insights into the molecular basis for its function that will inform efforts to identify this metabolic activity in the gut microbiota. Microorganisms in the human gastrointestinal tract and other anaerobic environments utilize choline as a source of carbon and energy. This process involves generation of TMA, an exclusively microbial metabolite (Figure 1a).4,5 While choline is an essential nutrient for humans,6 excess conversion of choline to TMA by the gut microbiota and its further oxidation to trimethylamine-N-oxide by liver enzymes is implicated in a number of diseases, including nonalcoholic fatty liver disease, atherosclerosis, and the metabolic disorder trimethylaminuria (fish malodor syndrome).7−9 TMA is also a carbon source for bacteria and is converted by archaea into the greenhouse gas methane in various marine habitats.10 We recently discovered the first genes responsible for anaerobic microbial choline metabolism.3 The choline utilization (cut) gene cluster, which is widely distributed among sequenced bacteria and found in many human gut isolates, encodes a predicted bacterial microcompartment© 2014 American Chemical Society
Received: February 16, 2014 Accepted: May 12, 2014 Published: May 22, 2014 1408
dx.doi.org/10.1021/cb500113p | ACS Chem. Biol. 2014, 9, 1408−1413
ACS Chemical Biology
Letters
Figure 1. Choline trimethylamine-lyase (CutC): a predicted glycyl radical enzyme (GRE) involved in anaerobic choline metabolism. (a) Microbial generation of TMA from choline and its subsequent processing in biological systems. (b) The choline utilization (cut) gene cluster and the biochemical role of predicted GRE CutC and GRE activase CutD. (c) Shared mechanistic hypothesis for GRE function and proposed activities for CutC and CutD. SAM = S-adenosylmethionine; dA = deoxyadenosine; Met = methionine.
typical of [4Fe−4S]2+ clusters: a broad peak at 380−420 nm that decreased in intensity upon reduction with excess sodium dithionite (NaDT) (Supplementary Figure S4).19 Analyses of CutD-bound iron and sulfide indicated the possibility of as many as two [4Fe−4S] clusters (8.4 mol of Fe and 7.6 mol of S per mole of CutD), although we cannot rule out adventitious binding of iron. The CutD extinction coefficient at 410 nm prior to reduction was ∼12.6 mM−1 cm−1, suggesting that not all iron−sulfur centers had been properly reconstituted ([4Fe− 4S]2+ ε410 ∼15 mM−1 cm−1).19 The EPR spectrum of NaDTreduced CutD supported this hypothesis. At 9 K, the spectrum contained an axial signal with g∥ of 2.045 and g⊥ of 1.94, which broadened at 40 K and did not change upon addition of excess SAM (Supplementary Figure S5). The g-values and temperature dependence of this signal further confirm the presence of [4Fe−4S]+ centers in CutD, while its intensity (0.4 spins per CutD monomer) is consistent with incomplete reconstitution. We examined the activity of NaDT-reduced CutD toward SAM in the absence of CutC using high performance liquid chromatography (HPLC) assays20 and observed generation of cleavage products 5′-deoxyadenosine and L-methionine (Supplementary Figure S6). Together, these analyses confirm the assignment of CutD as a radical SAM enzyme. We performed additional EPR experiments to investigate the reactivity of CutD toward CutC. Spectra of anaerobic reaction mixtures containing CutD, CutC, SAM, and NaDT taken at 77 K revealed a signal centered at g = 2.0037 and characterized by a 2-fold splitting of ∼1.46 mT (Figure 2a). The line shape of this signal and its g-value closely resemble those of the glycinecentered radicals from activated PFL21 and class III RNR.15 Under these conditions we activated 9.3% of the CutC dimers. Activation was not improved by employing catalytic amounts of CutD or by including choline in the reaction mixture. Use of the NADPH:flavodoxin reductase−flavodoxin system from E. coli22 decreased glycyl radical formation (
