Structural basis of carnitine monooxygenase CntA substrate specificity, inhibition, and intersubunit electron transfer

Microbial metabolism of carnitine to trimethylamine (TMA) in the gut can accelerate atherosclerosis and heart disease, these TMA-producing enzymes are therefore important drug targets. Here, we report first structures oxygenase CntA, an enzyme Rieske family. CntA exists a head-to-tail ?3 trimeric structure. The two functional domains (the catalytic mononuclear iron domains) located >40 Å apart same monomer but adjacent neighboring monomers. Structural determination subsequent electron paramagnetic resonance measurements uncover molecular basis so-called bridging glutamate (E205) residue intersubunit transfer. substrate-bound help define substrate pocket. Importantly, tyrosine (Y203) is essential for ligand recognition through ?-cation interaction with quaternary ammonium group. This between aromatic amine substrates allows us delineate subgroup oxygenases (group V) from prototype ring-hydroxylating involved bioremediation pollutants environment. Furthermore, discovery known inhibitors solve structure complex inhibitor, demonstrating pivotal role Y203 ?–? stacking inhibitor. Our study provides structural future drugs targeting this human gut. vast array cohabiting microorganisms impose discernible influence on well-being disease states. There considerable interest past decade investigate formation methylated amines health, particularly regard cardiovascular diseases nonalcoholic fatty liver (1Koeth R.A. Wang Z. Levison B.S. Buffa J.A. Org E. Sheehy B.T. Britt E.B. Fu X. Wu Y. Li L. Smith J.D. DiDonato Chen J. H. et al.Intestinal microbiota L-carnitine, nutrient red meat, promotes atherosclerosis.Nat. Med. 2013; 19: 576-585Crossref PubMed Scopus (2134) Google Scholar, 2Wang Klipfell Bennett B.J. Koeth R. Dugar B. Feldstein A.E. Chung Y.M. Schauer P. Allayee al.Gut flora phosphatidylcholine disease.Nature. 2011; 472: 57-63Crossref (2740) 3Cani P.D. Human microbiome: hopes, threats promises.Gut. 2018; 67: 1716-1725Crossref (413) 4Lynch S.V. Pedersen O. intestinal microbiome health disease.N. Engl. 2016; 375: 2369-2379Crossref (1008) 5Shreiner A.B. Kao J.Y. Young V.B. disease.Curr. Opin. Gastroenterol. 2015; 31: 69-75Crossref (527) Scholar). Dietary intake such as choline carnitine, both which prevalent diet, be processed by produce small (e.g., trimethylamine, TMA), enter vascular circulation leading hepatic oxidation oxide (TMAO). TMAO linked kidney diabetes, various forms cancers (6Zeisel S.H. Warrier M. Trimethylamine N-oxide, microbiome, disease.Annu. Rev. Nutr. 2017; 37: 157-181Crossref (142) 7Tang W.H. Hazen S.L. its diseases.Circulation. 135: 1008-1010Crossref (61) 8Jie Xia Zhong Feng Q. S. Liang Liu Gao Zhao Zhang D. Su Fang Lan al.The atherosclerotic disease.Nat. Commun. 8: 845Crossref (391) These metabolizing represent promising new targets (9Brown J.M. modulation Microbiol. 16: 171-181Crossref (150) 10Tang W.H.W. D.Y. metabolism, failure.Nat. Cardiol. 2019; 137-154Crossref (127) 11Schmidt A.C. Leroux J.-C. Treatments trimethylaminuria: where might heading.Drug Discov. Today. 2020; 25: 1710-1717Crossref (4) key microbial responsible TMA have only been identified relatively recently (12Craciun Balskus E.P. conversion requires glycyl radical enzyme.Proc. Natl. Acad. Sci. U. A. 2012; 109: 21307-21312Crossref (333) 13Zhu Jameson Crosatti Schafer Rajakumar K. Bugg T.D. Carnitine unusual rieske-type microbiota.Proc. 2014; 111: 4268-4273Crossref (149) 14Jameson T. Brown I.R. Paszkiewicz Purdy K.J. Frank Anaerobic microcompartments growth swarming Proteus mirabilis.Environ. 18: 2886-2898Crossref (22) Choline-TMA lyases belong large family proteins, attacking carbon–nitrogen (C-N) bond using species generated conserved glycine residue. choline-TMA lyase CutC has solved (15Bodea Funk M.A. Drennan C.L. Molecular C-N cleavage trimethylamine-lyase.Cell Chem. Biol. 23: 1206-1216Abstract Full Text PDF (37) 16Kalnins G. Kuka Grinberga Makrecka-Kuka Liepinsh Dambrova Tars Structure function bacterium Klebsiella pneumoniae.J. 290: 21732-21740Abstract (41) Scholar), aiding development (substrate analogs) attenuating (17Wang Roberts Zhu W. Gu Huang Zamanian-Daryoush Culley M.K. A.J. J.E. Krajcik al.Non-lethal inhibition production treatment atherosclerosis.Cell. 163: 1585-1595Abstract (549) 18Orman Bodea Campo A.M. Bollenbach Structure-guided identification molecule that inhibits anaerobic bacteria.J. Am. Soc. 141: 33-37Crossref (16) 19Roberts Hurd A.G. Gupta N. Skye S.M. Cody D.B. Barrington W.T. Russell M.W. Reed Duzan al.Development microbe-targeted nonlethal therapeutic inhibit thrombosis potential.Nat. 24: 1407-1417Crossref (167) 20Bollenbach Ortega Orman Discovery cyclic analog bacteria.ACS Lett. 11: 1980-1985Crossref (8) monooxygenase (CntA) associated reductase (CntB) (Fig. 1A) was originally Acinetobacter spp. subsequently found present range (13Zhu 21Jameson Doxey Airs Murrell J.C. Metagenomic data-mining reveals contrasting populations marine ecosystems.Microb. Genom. 2e000080PubMed belongs group non-heme-iron–containing typically multicomponent systems involving oxygenase, reductase, sometimes separate flavin cofactor (22Wackett L.P. Mechanism applications non-heme dioxygenases.Enzyme Microb. Technol. 2002; 577-587Crossref (80) component [2Fe-2S] center coordinated cysteine histidine residues (Fe) (23Barry Challis G.L. diversity rieske iron-dependent oxygenases.ACS Catal. 3: 2362-2370Crossref (111) 24Bugg Ramaswamy Non-heme dioxygenases: unravelling mechanisms enzymatic oxidations.Curr. 2008; 12: 134-140Crossref (164) A reduces pyridine nucleotides, generating electrons, ultimately transferred oxidation. archetypal oxygenases, also catalyze polyaromatic substrates; such, they environmental (24Bugg 25Perry C. de Los Santos E.L.C. Alkhalaf L.M. catalyse diverse reactions natural product biosynthesis.Nat. Prod. Rep. 35: 622-632Crossref several naphthalene dioxygenase biphenyl dioxygenase) Fe usually far subunit (>40 Å) effective transfer occur across interface subunits 26Karlsson Parales J.V. R.E. Gibson D.T. Eklund Crystal dioxygenase: side-on binding dioxygen iron.Science. 2003; 299: 1039Crossref (365) 27Shao Y.H. Guo L.Z. Y.Q. Yu Pang H.Q. Lu W.D. Glycine betaine monooxygenase, system, catalyzes oxidative N-demethylation Chromohalobacter salexigens DSM 3043.Appl. Environ. 84e00377-18Crossref (5) It becoming increasingly clear amines-oxidizing emerging clade distinct 28Daughtry K.D. Xiao Stoner-Ma Cho Orville Allen K.N. Quaternary demethylation: X-ray crystallographic, Raman, UV-visible spectroscopic analysis demethylase.J. 134: 2823-2834Crossref (32) 29Ertekin Konstantinidis K.T. Tezel Pseudomonas sp. BIOMIG1 converts benzalkonium chlorides benzyldimethyl amine.Environ. 51: 175-181Crossref (9) carnitine-degrading target, given increase plasma TMAO, However, no or description possible published. In study, high-resolution crystal without bound inhibitor-bound CntA. Structure, biochemical, (EPR) characterization mutants novel insights into mode inter-/intrasubunit work We set out baumannii successfully obtained apo protein form (2.1 Å, PDB 6Y8J) ligand-bound substrates, (2.0 6Y9D) ?-butyrobetaine (gBB; 1.6 6Y8S) (Table 1). observed 1B), cluster at opposing regions 44 1C). assembly homotrimer agreement analyses native gel Scholar) analytic filtration S1). superimposable average root-mean-square deviation 0.487 S1).Table 1X-ray data collection refinement statisticsNameCntA apoCntA + carnitineCntA gBBCntA+MMV12PDB code6Y8J6Y9D6Y8S6ZGPWavelength (Å)1.71.31.30.9Resolution (Å)40.1–2.05(2.12–2.05)81.42–1.97(2.04–1.97)39.78–1.63(1.69–1.63)78.64–2.01(2.08–2.01)Space groupP 63P 1 21 1P 1Unit cell91.6 91.6 82.96 90 12091.59 177.77 158.8 90.17 9091.16 91.16 81.47 12091.07 173.77 157.29 90.15 90Total reflections224,999 (13,603)1,180,725 (105,398)203,443 (9804)2,229,436 (218,489)Unique reflections24,714 (2443)353,116 (35,196)47,465 (4434)322,879 (32,095)Multiplicity9.1 (5.6)3.3 (3.0)4.3 (2.2)6.9 (6.8)Completeness (%)99.3 (99.2)98.8 (98.4)98.8 (93.4)99.4 (99.1)Mean I/sigma(I)7.41 (1.14)5.2 (1.21)6.8 (1.13)6.9 (1.13)Wilson B-factor (Å2)38312433R-merge0.189 (1.429)0.149 (0.958)0.134 (0.755)0.165 (1.783)R-meas0.200 (1.575)0.178 (1.168)0.152 (0.967)0.179 (1.929)R-pim0.064 (0.651)0.096 (0.660)0.070 (0.595)0.068 (0.733)CC1/20.994 (0.438)0.984 (0.638)0.982 (0.517)0.996 (0.698)CC?0.999 (0.781)0.996 (0.883)0.996 (0.825)0.999 (0.907)Reflections used refinement24,704 (2441)352,719 (35,160)47,464 (4434)321,994 (31,973)Reflections R-free1223 (101)17,582 (1828)2374 (221)16,243 (1504)R-work0.201 (0.429)0.207 (0.338)0.1704 (0.323)0.216 (0.386)R-free0.248 (0.530)0.243 (0.371)0.193 (0.339)0.254 (0.421)CC(work)0.958 (0.737)0.961 (0.805)0.963 (0.726)0.961 (0.799)CC(free)0.942 (0.821)0.953 (0.760)0.911 (0.768)0.948 (0.736)Number non-hydrogen atoms287435,752311736,701 macromolecules280334,344283234,901 ligands440831588 solvent6710002541212Protein residues34542243504328RMS(bonds) (Å)0.0080.0090.0070.009RMS(angles) (°)1.181.101.051.32Ramachandran favored (%)94.6795.3595.9194.76Ramachandran allowed (%)5.334.503.804.98Ramachandran outliers (%)0.000.140.290.26Rotamer (%)0.000.870.000.85Clashscore5.869.363.4113.20Average (Å2)55.9545.4435.7151.59 Macromolecules (Å2)56.2445.4034.9451.60 Center44.7441.3525.5648.08 FeN/A45.15176.4156.77 SubstrateN/A47.0257.75N/A InhibitorN/AN/AN/A69.75 Solvent (Å2)44.6939.2142.6144.22Number TLS groups9481044 Open table tab For all three structures, well defined 1D, Fig. S2A). structurally stachydrine demethylase Stc2 (28Daughtry 1,2-dioxygenase NdoB (26Karlsson dicamba DdmC (30D'Ordine R.L. Rydel T.J. Storek M.J. Sturman E.J. Moshiri F. Bartlett R.K. G.R. Eilers R.J. Dart Qi Flasinski Franklin S.J. Dicamba monooxygenase: dynamic exocyclic monooxygenation.J. Mol. 2009; 392: 481-497Crossref (30) S2B). active sites containing centers are, however, considerably varied S2B), reflecting catalyzed enzymes. octahedral geometry ligands (His213, His208) bidentate aspartate (Asp 323) well-documented His-His-Asp triad (31Hegg E.L. Que Jr., 2-his-1-carboxylate facial — motif iron(II) enzymes.Eur. Biochem. 1997; 250: 625-629Crossref (447) coordinating one face 1E). pair reduced Cys (Cys206, Cys209) near whereas corresponding formed disulfide bridge water thiocyanate [SCN]- anion occupy coordination sites, 2.2 2.3 away respectively 1E, S2C). Both positioned 3.7 below C? group, expected site hydroxylation SCN- O2 likely resulted stabilization cocrystalized substrates. purified ferrous (Fe2+) state, showing EPR signals either high-spin (S = 5/2) low-spin ½) ferric (Fe3+) despite purifying under aerobic conditions. confirmed addition nitric (NO) signal S ½ Fe2+-NO adduct 1F). contrast other nonheme enzymes, NO often led high-spin, 3/2 (32Wolfe M.D. Lipscomb Single turnover chemistry regulation activation 1,2-dioxygenase.J. 2001; 276: 1945-1953Abstract (158) plausibly suggests ion 0 state. similar previously reported heme-/nonheme-NO adducts (33Quaroni L.G. Seward H.E. McLean Girvan H.M. Ost W.B. Noble Kelly Price N.C. Cheesman M.R. W.E. Munro A.W. Interaction cytochrome P450 BM3.Biochemistry. 2004; 43: 16416-16431Crossref 34Cooper C.E. Nitric proteins.Biochim. Biophys. Acta. 1999; 1411: 290-309Crossref (433) revealed domain domain) subunit. As (12.2 apart) facilitated (E205, 2A) Electron NADH mediated mononucleotide (FMN)-containing CntB, activity To monitor pathway 2B) reductant via flavin-containing CntB continuous wave (cw)-EPR used. Purified EPR-silent 2C, black trace); ferredoxin readily detectable once added (red trace). spectrum characteristic [2Fe-2S]+1 [g tensor 2.031, 1.937, 1.899] resulting semiquinone gave 2.0015. Similar radicals monoamine oxidase (35Batie C.J. LaHaie Ballou D.P. Purification phthalate cepacia.J. 1987; 262: 1510-1518Abstract 36DeRose V.J. Woo J.C.G. Hawe W.P. Hoffman B.M. Silverman R.B. Yelekci Observation semiquinon resting state B nuclear double spectroscopy.Biochemistry. 1996; 11085-11091Crossref (31) Reduced simulated combination different spin states (97% 3% radical) 2), confirming (steps 1–3 2B). (blue trace) showed very weak anisotropic < 2.0 (g 2.011, 1.916, 1.757), indicating predominantly oxidized, diferric [2Fe-2S]2+ 2) (37Lee Simurdiak Reconstitution aminopyrrolnitrin N-oxygenase arylamine oxidation.J. 2005; 280: 36719-36727Abstract 38Rosche Fetzner Lingens Nitschke Riedel 2Fe2S centres 2-oxo-1,2-dihydroquinoline 8-monooxygenase putida 86 studied spectroscopy.Biochim. 1995; 1252: 177-179Crossref (17) Addition oxidizing agent (H2O2) minimal effect overall S3A); dithionite S3B). appears capable (carnitine) H2O2 absence CntB/NADH S3A), consistent peroxide shunt-mechanism (39Wolfe Hydrogen peroxide-coupled cis-diol 278: 829-835Abstract (96) 40Neibergall M.B. Stubna Mekmouche Münck dependent cis-dihydroxylation benzoate fully oxidized 1,2-dioxygenase.Biochemistry. 2007; 46: 8004-8016Crossref (62) order flow recorded (magenta 2, S4). Thus, reduction cross-subunit (step 4 2B).Table 2Spin-Hamiltonian parameters model spectra shown 2CSamplesEPR signalSpecies%g1g2g3gaveAs-isolated CntA2.01051.91571.75741.8945As-isolated CntBNo detectedCntB+NADH[2Fe-2S]+1specA (reduced domain)932.03141.93721.89881.9558Flavin radicalspecB (Flavin radical)72.00152.00152.00152.0015CntA+CntB+NADH[2Fe-2S]+1 CntBspecA domain)612.03131.93761.89791.9556Flavin radicalspecB22.00102.00342.00392.0028[2Fe-2S]+1 CntAspecC center)372.00791.91531.75421.8924CntA+CntB+NADH+Carnitinean organic radicalspecB342.00002.00352.00372.0024[2Fe-2S]+1 CntAaThis may contain overlapping derived center; plausible intermediates ferric-(hydro)peroxy high-valent iron(V)-oxo species.specC662.00711.90771.79861.9045E205A+CntB+NADH+Carnitine[2Fe-2S]+1 CntBspecA1402.03381.93971.88681.9534[2Fe-2S]+1 CntBspecA2202.04491.94931.90371.9660Flavin radicalbThis overlay unidentified radical.specB82.00122.00402.00402.0031[2Fe-2S]+1 species.specC322.00701.90771.78961.9014a species.b radical. next sought confirm critical E205 suggested 5 bridges M