Converging on a Mechanism for Choline Degradation

Home , Ethanolamine, Glycerol dehydratase

COMMENTARY

Converging on a mechanism for choline degradation

Christopher J. Thibodeauxa and Wilfred A. van der Donka,b,c,1 aInstitute for Genomic Biology, bHoward Hughes Medical Institute, and cDepartment of Chemistry, University of Illinois, Urbana, IL 61801

n PNAS, Craciun and Balskus (1) combine biochemical intuition with I modern genome mining techniques to discover the elusive enzyme re- sponsible for catalyzing the degradation of choline (1, Scheme 1). Choline is an essential nutrient in higher organisms that is required for the biosynthesis of the neu- rotransmitter acetylcholine and the head group of several phospholipids, and that serves as a source of the methyl groups for methionine and S-adenosylmethionine (2). In humans and other mammals, choline is catabolized to trimethylamine (TMA; 2, Scheme 1) by symbiotic gut microbes, Scheme 2. Putative chemical mechanism of ethanolamine degradation by the adenosylcobalamin- and irregularities in choline and TMA dependent enzyme, ethanolamine ammonia lyase (EAL). metabolism have been linked to liver and cardiovascular diseases, atherosclerosis, sponsible for choline degradation in the with a protein-derived, Cα-centered glycyl and deficiencies in fetal brain develop- anaerobic organism Desulfovibrio de- radical. Furthermore, a homology model ment (3–5). TMA derived from choline sulfuricans (an organism that metabolizes of CutC suggested similarities in its ac- is also a substrate for methanogenesis by choline to TMA but whose genome does tive site architecture with another gly- marine microorganisms and, as such, not encode EutBC homologs), Craciun cine radical enzyme, the B12-independent contributes to global production of the and Balskus mined the genome for the glycerol dehydratase (9), which catalyzes eut 7 greenhouse gas methane. presence of genes involved in formation of 3-hydroxypropanal ( , acetaldehyde processing. Scheme 1) from glycerol (8, Scheme 1). To identify the unknown choline fi degrading enzyme, which has eluded This approach led to the identi cation Bioinformatic analyses demonstrated that cut researchers for more than a century, of the choline utilization ( ) gene clus- CutC homologs form a distinct clade of the authors postulated that the conversion ter, which contains several tightly clustered glycyl radical enzymes and that they are of choline to TMA and acetaldehyde homologs of eutE, eutG, and other eut encoded in the genomes of organisms that (3, Scheme 1) may share mechanistic genes. Interestingly, within this gene clus- are known to ferment choline as well as in similarities to the well-studied catabolism ter are also encoded a predicted glycyl human commensural bacteria and marine 4 radical enzyme (cutC) and a glycyl radical microorganisms. On the basis of these of ethanolamine ( , Scheme 1) to ammo- fi nia and acetaldehyde by bacterial enzymes activating protein (cutD), enzymes not ndings, the authors postulate that CutC is encoded in the ethanolamine utilization known to catalyze C-N bond cleavage. acholineTMAlyaseenzymethatcatalyzes (eut) gene cluster (6–8). In ethanolamine Subsequent genetic disruption of the conversion of choline to TMA using radical degradation, C-N bond cleavage is carried cutC gene in the anaerobic choline user chemistry in a manner analogous to the out by the adenosylcobalamin (AdoCbl)- Desulfovibrio alaskensis G20, and heterol- EutBC-mediated C-N bond cleavage of dependent enzyme, ethanolamine ammonia ogous expression of the cutC and cutD ethanolamine. lyase (EAL), encoded by the eutBC genes genes from D. alaskensis G20 in Escherichia The well-studied chemical mechanisms (6). After the EAL-catalyzed cleavage of coli confirmed the essential role of these of EAL (10) and the B12-independent the C-N bond of ethanolamine, the genes in the conversion of choline to glycerol dehydratase (9, 11) can serve as a acetaldehyde product is converted to TMA. In addition, whole-cell electron basis for a mechanistic model of catalysis ethanol (5, Scheme 1) and acetyl-CoA paramagnetic resonance studies showed by choline TMA lyase. In EAL (Scheme 2), (6, Scheme 1) by alcohol dehydrogenase that D. desulfuricans cells grown on media substrate binding triggers homolytic (EutG) and aldehyde oxidoreductase containing choline possess significant cleavage of the Co-C bond of the AdoCbl (EutE) enzymes, respectively. In a clever quantities of a paramagnetic species whose coenzyme, generating cob(II)alamin and approach to identify the gene cluster re- spectroscopic properties were consistent the transient, reactive 5′-deoxyadenosyl radical (5′-dAdo•). 5′-dAdo• subsequently abstracts a hydrogen atom (H•) from C1 of ethanolamine to generate the well- characterized substrate radical, 9 (Scheme 2) (12). The exact chemical mechanism from this point remains unclear, but several computational studies agree that the low- est energy reaction coordinate involves a 1,2-migration of the amine group to

Author contributions: C.J.T. and W.A.v.d.D. wrote the paper. The authors declare no conﬂict of interest. Scheme 1. Mechanistic similarities in the catabolism of choline, ethanolamine, and glycerol; in each See companion article 10.1073/pnas.1215689109. case, the bonds highlighted in red are cleaved by enzyme-mediated radical chemistry that results in a 1,2- 1To whom correspondence should be addressed. E-mail: migration. [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1219534110 PNAS Early Edition | 1of2 Downloaded by guest on October 3, 2021 substrate radical to give the resonance stabilized product radical (17, path b, Scheme 3). In the case of EAL, however, both experimental and computational studies have shown that the sluggish re- activity of stable product radicals similar to 17 (Scheme 3) hinder regeneration of the active form of the AdoCbl coenzyme and act instead as suicide inhibitors (13, 20). Clearly, further mechanistic, structural, and computational studies of choline TMA lyases will be required to better in- terrogate their chemical mechanism and reaction energetics, to assess the feasibility of the key 1,2-TMA group migration step (15 → 16, Scheme 3) and to define the roles of active site amino acids in this biologically important enzymatic transformation. Scheme 3. Whereas many details of the EAL and Putative chemical mechanisms of choline degradation by the glycyl radical enzyme, choline choline TMA lyase-catalyzed reactions trimethylamine lyase (CutC). Amino acids are numbered according to their positions in CutC from D. desulfuricans. have not yet been elucidated, the C-N bond cleavage reactions catalyzed by these enzymes illustrate an interesting example generate the carbinolamine product radi- a glycyl radical on CutC (13,Scheme3). of convergent evolution—whereby struc- cal (10, Scheme 2) (13, 14), perhaps via As in other glycyl radical enzymes, CutC turally unrelated enzymes have evolved a cyclic transition state such as 11 (Scheme is expected to use an active site thiyl to catalyze similar chemical transforma- 2). Partial protonation of the migrating radical (14, Scheme 3) for the initial tions. Interestingly, this is not the first amine group and partial deprotonation of H• abstraction from substrate (17–19). reported example of convergent evolution the hydroxyl group at C1 by active site After formation of the substrate radical between AdoCbl- and glycine radical- 15 amino acids likely play critical, synergistic ( , Scheme 3), the trimethylamino group dependent enzymes: this phenomenon has roles in lowering the energy of the transi- at C2 may undergo a 1,2-migration to been observed for ribonucleotide reduc- tion state leading to migration (13, 15). C1 (path a) to give a carbinolamine radical 16 tases and glycerol dehydratases (19). Thus, Consistent with this hypothesis, a recent X- ( , Scheme 3) analogous to the proposed this type of convergent evolution is be- ray crystal structure of EAL from E. coli product radical in the EAL-catalyzed 10 ginning to emerge as a common theme for revealed a constellation of conserved acid/ reaction ( , Scheme 2). According to these radical enzymes and could perhaps base and polar amino acid residues that the glycerol dehydratase homology model fl seemed poised to guide the amine group proposed by Craciun and Balskus (1), re ect an adaptation by microbial or- along a 1,2-migration trajectory (16). After this migration may be facilitated by ganisms to bypass the different suscepti- amine migration, the reactive carbinol- Glu493-mediated partial deprotonation of bilities of radical enzymes to irreversible amine radical (10, Scheme 2) abstracts the C1-OH group and perhaps by elec- inactivation by molecular oxygen. Re- a hydrogen atom from 5′-dAdo to form trostatic interactions between the migrat- gardless of what pressures have shaped carbinolamine (12,Scheme2)andre- ing TMA group and the conserved Asp218 the evolution of choline TMA lyases, it is generate the resting state of the AdoCbl residue (1, 9, 11). Regeneration of the clear that they constitute a unique class coenzyme. To complete catalysis, the cysteine thiyl radical and elimination of of enzymes that have expanded the already amino group of the carbinolamine is TMA would complete the CutC catalytic impressive catalytic repertoire of glycine eliminated to give the reaction products cycle. In line with a recent computational radical enzymes (17–19). With their study, ammonia and acetaldehyde. study of the B12-independent glycerol de- Craciun and Balskus have answered a Extrapolating our current understanding hydratase (11), another potential model long-standing question regarding choline of EAL and glycyl radical enzymes to for CutC catalysis could involve direct catabolism and opened up a unique choline TMA lyase, CutD will first generate elimination of the TMA group from the branch of glycyl radical enzymology.

1. Craciun S, Balskus EP (2012) Microbial conversion of cho- of choline by extracts of Vibrio cholinicus. JBiolChem 14. Semialjac M, Schwarz H (2002) Computational exploration line to trimethylamine requires a glycyl radical enzyme. 235:3592–3596. of rearrangements related to the vitamin B12-dependent Proc Natl Acad Sci USA, 10.1073/pnas.1215689109. 9. O’Brien JR, et al. (2004) Insight into the mechanism ethanolamine ammonia lyase catalyzed transformation. 2. Stead LM, Brosnan JT, Brosnan ME, Vance DE, Jacobs RL of the B12-independent glycerol dehydratase from JAmChemSoc124(30):8974–8983. (2006) Is it time to reevaluate methyl balance in humans? Clostridium butyricum: Preliminary biochemical and 15. Sandala GM, Smith DM, Radom L (2010) Modeling the – – Am J Clin Nutr 83(1):5 10. structural characterization. Biochemistry 43(16):4635 reactions catalyzed by coenzyme B12-dependent enzymes. fi 3. Dumas ME, et al. (2006) Metabolic pro ling reveals a 4645. Acc Chem Res 43(5):642–651. contribution of gut microbiota to fatty liver phenotype 10. Bandarian V, Reed GH (1999) Ethanolamine ammonia- 16. Shibata N, et al. (2010) Crystal structures of ethanolamine in insulin-resistant mice. Proc Natl Acad Sci USA 103(33): lyase. Chemistry and Biochemistry of B , ed Banerjee R 12 ammonia-lyase complexed with coenzyme B12 analogs 12511–12516. (Wiley-Interscience, New York), pp 811–833. and substrates. J Biol Chem 285(34):26484–26493. 4. Wang Z, et al. (2011) Gut flora metabolism of phospha- 11. Feliks M, Ullmann GM (2012) Glycerol dehydratation 17. Frey PA (2001) Radical mechanisms of enzymatic catalysis. tidylcholine promotes cardiovascular disease. Nature by the B12-independent enzyme may not involve the Annu Rev Biochem 70:121–148. 472(7341):57–63. migration of a hydroxyl group: A computational study. 18. Selmer T, Pierik AJ, Heider J (2005) New glycyl radical 5. Zeisel SH, da Costa KA (2009) Choline: An essential J Phys Chem B 116(24):7076–7087. enzymes catalysing key metabolic steps in anaerobic nutrient for public health. Nutr Rev 67(11):615–623. 12. Bender G, Poyner RR, Reed GH (2008) Identification of – 6. Garsin DA (2010) Ethanolamine utilization in bacterial the substrate radical intermediate derived from etha- bacteria. Biol Chem 386(10):981 988. pathogens: Roles and regulation. Nat Rev Microbiol 8(4): nolamine during catalysis by ethanolamine ammonia- 19. Buckel W, Golding BT (2006) Radical enzymes in anae- – 290–295. lyase. Biochemistry 47(43):11360–11366. robes. Annu Rev Microbiol 60:27 49. 7. Bradbeer C (1965) The clostridial fermentations of choline 13. Wetmore SD, Smith DM, Bennett JT, Radom L (2002) 20. Abend A, Bandarian V, Reed GH, Frey PA (2000) Iden- and ethanolamine. 1. Preparation and properties of Understanding the mechanism of action of B12- tification of cis-ethanesemidione as the organic radical cell-free extracts. JBiolChem240(12):4669–4674. dependent ethanolamine ammonia-lyase: Synergistic derived from glycolaldehyde in the suicide inactivation 8. Hayward HR (1960) Anaerobic degradation of choline. interactions at play. J Am Chem Soc 124(47):14054– of dioldehydrase and of ethanolamine ammonia-lyase. III. Acetaldehyde as an intermediate in the fermentation 14065. Biochemistry 39(20):6250–6257.

2of2 | www.pnas.org/cgi/doi/10.1073/pnas.1219534110 Thibodeaux and van der Donk Downloaded by guest on October 3, 2021