Purinyl-Cobamide Is a Native Prosthetic Group of Reductive

Home , Cobalamin biosynthesis, Reductive dehalogenases

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. C E NATURE CH *e-mail: of Research andGraduate U Sciences (J T I 1 obligate of growth and rates dechlorination reductive impact does cobalt( reac tive the restoring for transfer electron or catalysis in role direct atom cobalt active redox- the from distant anduncoordinated are adenine, and DMB base-off configurations, indicating that their respective lower bases, B coenzyme groups from ID tion of carbon–halogen bond cleavage In either case, the cobalt( (RDases) dehalogenases reductive corrinoid-dependent by lyzed capable of generating the cobalt( B vitamin by abiotically mediated be can reactionsdehalogenation Reductive loop nucleotide the to connected bases lower known 16 group5 a or nucleotide loop the in chain side the case, documented one in and, base, lower the ligand, upper the in exist structure (Cba) cobamide Variationsin ( base lower the as (DMB) 5,6-dimethylbenzimidazole and Co upper an as group cyano artificial the carries balamin) is connected to the corrin ring corrin ring, and a lower Co amides) consist of an upper Co ment and reductive dehalogenation activities generate to manipulate functionalitiesofmicrobiomes. opportunities enzyme modulate effectively can bases lower that realization the and structure cobamide novel a of discovery the pathways, metabolic essential in keysteps catalyze enzymes cobamide-dependent that Given bacteria. organohalide-respiring otrophic, different lower bases are not functionally equivalent, and purinyl-Cba elicits different physiological responses in corrinoid-aux of tetrachloroethenedehalogenasesreductiveactive catalytically in group Codemonstratingthat by function biological a purine unsubstituted dehalogenases. This study reports the biosynthesis of a new cobamide with unsubstituted purine as the lower base and assigns and anthropogenic organohalogens, cobamides are the requisite prosthetic groups for carbon–halogen bond-cleaving reductive natural of cycling global the forprocess biological vital respiration,a organohalide In life. of domains all in functions chemical Cobamides such as vitamin B A Jun Yan of reductive dehalogenases P D nstitute ofApplied ennessee, Knoxville, SA. lizabeth epartment ofMicrobiology,epartment ndrew T eety eovd Ds cytl tutrs f pdA (PDB NpRdhA of structures crystal RDase resolved Recently ennessee, Knoxville, 4RA urinyl-cobamide isanative prosthetic group 7 Sulfurospirillum multivorans D orrinoids are the most complicated organometallic cofactorscomplicatedorganometallic most the are orrinoids including methyl group transfer, carbon skeleton rearrange skeleton transfer,carbon group reactionsmethyl including biochemical essential catalyze to biology in used epartment of epartment [email protected] S ) from ) E IB 12 1 MIC T – I n is nlg i te rsne f srn reductant strong a of presence the in analogs its and S), ) state. Contrary to expectations, lower-base structure lower-base expectations, to Contrary state. ) 5 ` -deoxyadenosyl moiety as the upper ligand and oneandupperligandof the asmoiety -deoxyadenosyl Quaile *, MengBi A A O L BIOLOGY

3 E Nitratireductorpacificus ak Ridge . Physiologically functional cobamides use a methyl E dwards cology, 7, C T 8 ennessee, 12 Teeoe te oe bs i nt sind a assigned not is base lower the Therefore, . hemical E T n norpseudo-B and ducation, 7 ennessee, ,

N | ADV N I ) supernucleophile is critical for the initia C ational U annan Jiang hinese Academy ofSciences, Shenyang, A 1 or 7 , E niversity of base as part of the nucleotide loop that A

A ngineering andApplied U NCE ONLINE PUBLIC [email protected] 2 . For example, vitamin B B U SA.

&Frank llen KBourdon 12 U I L ligand, a central cobalt-containing niversity of ) supernucleophile are structurally conserved, cobalt-containing tetrapyrrole biomolecules that have essential bio aboratory, SA. demonstrate that their prosthetic 4 1 B . Complete corrinoids (i.e., cob iosciences 10 6 D T . ennessee, Knoxville, epartment of epartment and PceA (PDB ID PceA(PDBand 12 , respectively, are in the the in are respectively, , 3 O T E – ennessee, Knoxville, 5 ak Ridge, Löffler , 8 A D

TION | ivision,

, Yi Yang C 6 4 B hemistry, or can be cata www.nature.com/naturechemicalbiology T iosystems ennessee, 1 O

, , 12 3 ak Ridge A (cyanoco – 2 5 T ( , bigail B 8 ennessee, Fig. Fig. – ligand 4UR U 10 3 , 9 niversity of T E * L , Yongchao Yin U ennessee, ngineering andSoilScience, iaoning, N 1 1 SA. 0 ational ). ). 5 ------) . T PU 6 U Farmer D SA. BLI ( predominantly generate and group, pros a thetic as cobamide for requirement strict a have RDases (PCE) solvents chlorinated as such con taminants groundwater including organohalogens, of dehalogenation subsurfaceenvironments,in contribute wherethey reductive the to anaerobic,commonversatile,Gram-positive metabolically bacteria discovered in1989(ref. of phenolyl-cobamide in phenol ture, struc recent most the with bacteria, anaerobic from mostly tified, lacking. To date, 16 naturally occurring lower bases have been iden is understanding mechanistic the however,organisms; host the to bases can affect enzyme activity and cause substantial consequences thoxybenzoate as substrates respectively, severely inhibiting growth with methanol or 3,4-dime cobalamin, or (5-MeBza-Cba) 5-methylbenzimidazolyl-cobamide non-nativeformationof the in results medium the to DMB or Bza Wood–Ljungdahlpathway the using tion assimila carbon and conservation energy for methyltransferases gen homoaceto the in utilization substrate affects also structure base activity adenine) 5-OHBza; dechlorination 5-hydroxybenzimidazole, reductive (e.g., of loss complete cause or compromise Bza) structures lower-base other 5-OMeBza;benzimidazole, 5-methoxybenzimidazole, (e.g., growth with cobamides and base, lower the as (5-MeBza) 5-methylbenzimidazole or DMB ing and activity mccartyi corrinoid-auxotrophic organohalide-respiring, epartment of epartment c P C) n iognc hoie s products as chloride inorganic and DCE) L .R. T U S 2 aboratory, oronto, ebr o te genus the of Members Key SA. a H C prms ovata Sporomusa ED -purinyl-cobamide (purinyl-Cba) is the native prostheticnative the is (purinyl-Cba)-purinyl-cobamide hina. 9 L D aboratory of O ( 6 epartment of epartment , T N 3 oronto, Dhc P C Desulfitobacterium hafniense Desulfitobacterium LI 1 C O Dhc enter for , Burcu o-Hsiang Wang

NE: 6 hemistry, ak Ridge, strains ) growth require the addition of cobamides carry cobamides of addition the require growth O U NO ntario, P niversity of ollution E C VE nvironmental wih ss hnlc cobamide-dependent phenolic uses which , U S T ¸ ivil and ennessee, niversity of 1 ims MB 9, 3 1 C 0 ). Mxmm eutv dechlorination reductive Maximum . anada. ER 2017 | 1 ¸ E 2 . These findings indicate that the lower ir Desulfitobacterium cology and E T nvironmental 3 ennessee, Knoxville, , 9 U , 8 B 7 T B SA. S

ennessee, Knoxville, redesen iotechnology,

hawn D

, OliviaMolenda OI 5 Joint E 14, : 10.1038/N prms ovata Sporomusa nvironmental 1 1 2 5 C . Cobamides featuring Cobamides . . The addition of 5-Me of addition The . Te tetrachloroethene The . E I nstitute for R enter for ngineering, ARTICLE cis 1 Campagna 6 Te characterized The . -1,2-dichloroethene U ( niversity of T Dsf ennessee, CH Dehalococcoides 10, I E nterdisciplinary ) are strictly strictly are ) T E 1 ngineering, B ennessee, 1 U

MBIO Te lower The . iological niversity ( Fig. U 7 .251 , 6 SA.

, 1 ), ------1

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. ARTICLE 2 Dsf the for formula molecular possible a information, mass on Based naturalcobamide.known anycurrently from distinct is that amide organohalide-respiring the that suggested form cyanated their in cobamides occurring naturallyof a yet-unidentified lower Comparison base. to masses calculated the nant predomi the that indicated form) monocyano the in Da (1,016.12 molecular the ofmass measured the between Da 312.42 base), of difference lower mass the a as 2-methylsulfonyladenine (with Da 425.31 to base) lower the as phenol (with 306.2 from ranges cobamides ral the of mass ular ( respectively forms, ion 1,329.54 and 1,351.52 corresponding to their [M+H] hafniense D. from corrinoids B vitamin [M+Na] represented the Fig.3a [M+H] ions corrinoid intact to corresponding with peaks base B vitamin of tions may represent Cbi precursors ( 15.95 min) similar to those of Cbi, suggesting that these minor and frac (15.74 times retention and spectra absorption showed predominant rinoid the after eluted that fractions minor Two ( maximum absorption nm 355 a with spectrum Cbi the from distinct was butmaximum,absorption 361-nm includingthe ( rinoid predominant the of spectrum (Vis) ing that the dicyano-Cbi as min Supplementary Fig. 15.63 1b and monocyano-Cbi as min 8.77 at eluted which cobinamide (Cbi;ofdifferentthat from was time tion Dsf 2 Fig. standards, Cba seven for times retention of range the (i.e., areas at 361 nm of the total fractions eluting between 10 and 18 min Results Supplementary times ( min retention 11.79 with of corrinoids candidate three of presence the (i.e., cultures pure upper the as group cyano a example, (for form cyanated the of analysis HPLC Discovery andstructure ofanovel cobamide RE unsubstituted naturally purine. of to diversity function biological a assign the and structures cobamide occurring expand obtained results The groups. prosthetic corrinoid of characterization rapid for approach vative inno an illustrating separation, electrophoretic following RDase PCE active catalytically in achieved was purinyl-Cba of detection Direct bacteria. organohalide-respiring corrinoid-auxotrophic ent Dsf ( cobamide(s) remains elusive tionand attachment, but identity the of lower the of base native activa base lower and biosynthesiscobinamide for required genes that corroborates analysis of these capable are that indicating corrinoids, exogenous without PCE-dehalogenating 1

) as the native cobamide produced in axenic PCE-dechlorinatingaxenic producedin nativecobamide the as ) Here we report the discovery and characterization of purinyl-Cba Mass spectra of the the of spectra Mass S cobamide was deduced as C retenThis culturesfraction. min 11.79 associatedthe waswith differ in functionality distinct its demonstrate and cultures UL Dsf a , ) showed that the predominant corrinoid produced in the the in produced corrinoid predominant the that showed ) TS b 1 corrinoid was a cobamide carrying an carrying cobamide a was corrinoid ). The base peaks with peaks base The ). sbtnily vrapd ih ht f iai B vitamin of that with overlapped substantially ) 12 Dsf rsetvl. h ms seta f h predominant the of spectra mass The respectively. , Dsf tan 5, hwn bs pas with peaks base showing Y51, strain 12 B tnad band sn L–S ipae strong displayed LC–MS using obtained standard strains produced a distinct corrinoid. The UV-visible de novo de ligand) of corrinoid extracts from PCE-grown from extracts corrinoid of ligand) 1 corrinoid and the calculated molecular mass of Cbiofmass molecular calculated corrinoid andthe , 57 mn ( min 15.74 ), Dsf m/z A Dsf -ribazole-5 strains JH1, Viet1 and PCE1 matched that of that matched PCE1 and Viet1 JH1, strains values of 1,329.54 and 1,355.58, respectively, 1,355.58, and 1,329.54 of values Dsf strains Y51, JH1, Viet1 and PCE1) revealed revealed PCE1) and Viet1 JH1, Y51, strains corrinoid biosynthesis corrinoid ) and any of the cobamide standards, indicat , + Supplementary Fig. 3c Fig. Supplementary ue utrs rw n eie medium defined in grow cultures pure ion form of the strain Y51 corrinoid and corrinoid Y51 strain the of form ion Supplementary Fig. 1a Fig. Supplementary . hafniense D. Dsf 1 8 . ` tan pses h cmlt st of set complete the possess strains -phosphate ( -phosphate 2 m/z 59 ad 59 mn ( min 15.95 and ) Dsf H 82 valuesofand 1,351.52 1,377.56 strains produce a unique cob unique a produce strains Supplementary Fig. 2 CoN . hafniense D. tan 5 criod and corrinoid Y51 strain 16 O A 14 -RP) moiety in natu in moiety -RP) 1 P. 7 . Genome sequence Genome . – + A ). Integrated peak Integratedpeak ). ( e tan 5 cor Y51 strain -RP moiety with moiety -RP ). As the molec the As ). Supplementary 3 + ( ) m/z and [M+Na] NATURE i. 2 Fig. Dsf aus of values Fig. 2 Fig. ). Dsf strains 12 a CH ( 4 cor and – 1 E Dsf Dsf 1 b 0 MIC NA 1 0 ). ), 9 ------+ ; ;

A L T

having ( trum UV-Visspec time, retention HPLC by determined as cobalamin, formationof the in resulted Y51 strain approachwithbiosynthesis cobamide non-native a of synthesis the 25 of ence pres the in acceptor electron an as PCE with cultures Y51 strain novel this of structure namide whether demethylation or any other modification exists in the cobi multivorans ( values for the base peaks of the [M+H] the of peaks base the for values The structure. base lower unknown the in atoms nitrogen of with experiments conducted we characteristics, structural these nitrogen 14 = atoms) 2 or phenol derivatives+ (12 + 0 (12 = 12 nitrogen Bza atoms).atoms), Based on nitrogen 17 = 5 + 12 guanine; or adenine example, (for nucleobases carry that cobamides from an unsubstituted with purine as the lower base (12 + 4 = cobamide16 nitrogen atoms) a distinguishes atoms nitrogen of number the Thus, atoms. nitrogen 17 to 12 possess cobamides occurring rally natu base, lower the on atoms.Depending nitrogen 12 of number constant a carries form cyanated the in skeleton acid cobinic The Confirmation ofthenovel lower basestructure (DMB)]), is which suggestive of unsubstituted purine. Dsf the in tion,weestimated massmolecular oflower compound the the base informa this on Based modifications.unusual any possess not did confirmed that the new cobamide natively synthesized in the of functionality supported groups prosthetic different the that indicated Bza-Cba the producing cultures Y51 PCE-to- similar Thus, suggested have studies prior as skeleton acid cobinic the of bly ( Bza received 4 Fig. that cultures Y51 strain in sively 6-methylbenzimidazole; 5- C structures are found inthe found innaturally occurring cobamides. Figure 1|Cobamide general structure andthe16known lower bases 2-methylsulfinyladenine; 2-S 2-methyladenine; 2-SMeAde, 2-methylmercaptoadenine; 2-S benzimidazole; 5-Me BIOLOGY SupplementaryFig. 3f URE 176, andthe O n h nrtp criod rdcd by produced corrinoid nor-type the In cobamide) – [1,355.58 ([M+H] [1,355.58 – cobamide) Me ). Guided cobamide biosynthesis does not change the assem change the not does biosynthesiscobamide Guided ). B Fig. 2c Fig. Dsf

m/z za, 5-methoxybenzimidazole; 5- | ADV C HEM cobamide to be 120.15 (1,329.54 ([M+H] (1,329.54 120.15 be to cobamide M values of 1,355.58 ([M+H] 1,355.58 of values , C176 of the side chain is demethylated A C M DMB. The addition of DMB was shown to trigger trigger to shown was DMB of addition The DMB. M , NCE ONLINE PUBLIC o d Low A ), and mass spectral features with two strong peaks peaks strong two withfeatures spectral mass and ), lower base.5- IC er base D.hafniense B za, 5-methylbenzimidazole; 2-MeAde, AL Dsf D c DCE dechlorination performance in strain in performance dechlorination DCE M C ). Similarly,). ( Bza-Cba we detected cA Ds. olciey tee findings these Collectively, RDase. PceA BI o O B B , 5,6-dimethylbenzimidazole; 2 upperligand, thesidechainatposition MeAde, 2-methylsulfonyladenine. OLO O Nor Co C Vi A Dsf 2-S 5-OHBza strain Y51 to determine the number the determine to Y51 strain oenzyme B TION | Adenine Bz Dsf tamin B H mmon-type corrinoid:R=CH MeAde -type corrinoid:R= a5 B aie oaie cblmn or cobalamin, cobamide, native za, 5-hydroxybenzimidazole; cobamide, we grew we cobamide, G + 12 O www.nature.com/naturechemicalbiology

Y of cyanocobalamin) – 146.19 – cyanocobalamin) of V : X=CN,RCH 12 Me-6-Me 2- Hypo ariations incobamide : X=5’-deo

SOMeAde 5-OMeBza D -MeBza + 12, ) and 1,377.56 ([M+Na] 1,377.56 and ) O + xanthine and [M+Na] and 2 I 0 : . This guided cobamide guided This .

1 0 H . xy 1 5-OMe-6-MeBza B 2- 15 0 adenosyl, R=CH 3 za, 5-methoxy- SO N isotope-labeling N 3 Guanine Phenol 8 2 3 MeAd / 3 Supplementary Sulfurospirillum ( N Fig. B C + e za, O of cyanated of H D. hafnienseD. + MeAde, E ion forms ion Naphthimidaz Dsf M 1 p 2- 5 3 -C ). To test DMB B MeAde ) exclu) re strains I sol O . 2 m/z 20, 5 ole 2 1 + 1 2 ------) .

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. NA NATURE CH the protons,but neighboring between couplingspin–spin revealed the with B vitamin of portion DMB the with associated primarily was which p.p.m.), (9.0–6.0 region aromatic the in occurred shifts) chemical native ( spectra native the using ments experi spectroscopy NMR 2D and 1D conducted we base, lower stituted as lower the native inthe purinebase served unsub that evidence additional provided analysis MS by followed the for 1,329.54 of [M+H] measured the from different is which 1,345.29, of mass molecular calculated a has form cyanated its in thine-Cba nativein a cobamideof Hypoxanthine, a purine derivative with 4 N atoms, is the lower base 16–12). indi (i.e., atoms nitrogen ligand), 4 contains base CN lower the upper that cating the in nitrogen unlabeled one plus (15 native the in atoms nitrogen of number total ( increase 1,351.52 to 1,366.46, respectively, corresponding to a 14.94 Da mass the of protons onthestructure to thecorresponding vitamin superimposed Dsf [M+H] spectra of wavelengths ( amendment. non-native cobamide produced instrain Y51 cultures with25 Dsf (250–600 nm)ofcobinamide (cbi), vitamin ( 5- 5- each) were norpseudovitamin Y51 basedon361 nmpeakarea integration. 11.79 minaccounted for 86%ofthetotal corrinoids produced by strain ( native corrinoids produced by Desulfitobacterium Figure 2|Spectrophotometric andstructural features of 1 1 a e c 12 0 ), 15.74 min( O O Relative abundance (%) A361 (mAU)

cobamide contains four corrinoid. ( A361 (mAU) Hmncer orlto setocp (OY experiments (COSY) spectroscopy correlation Homonuclear . To confirm unsubstituted purine as a new naturally occurring occurring naturally new a as purine unsubstituted confirm To ), 15.02 min.mA 100 0 0 0 Me H 2 2 2 0 3 3 3 50 2 3 1 1 1 1 1,320 T 10 0 10 B za- URE + B Dsf 15 B and[M+ za- 1,329.54 N-labeled cobamide shifted from 1,329.54 to 1,344.48 and 1,344.48 to 1,329.54 from shifted cobamide N-labeled 12 C i. 2 Fig. 15 standard (blueline). 45 E Dsf Fig. 2 Fig. cobamide ( ∆ ba ( N MIC C =14.9 12 12 1 D ba ( -labeled andunlabelednative L Retention time(min Retention time(min oaie n vtmn B vitamin and cobamide 1,340 c 1 C otted linesin 2 H max A 8 ) H 89 ), and15.94 min( 1,344.4 4 L BIOLOGY ), 13.13 min;5-Me HEM 6 f N N e ) ofacobinamide oracobamide, respectively. ( o atetc iai B vitamin authentic of ) ), 13.88min; ). Based on this mass shift, we calculated that the the that calculated we shift, mass this on Based ). PLC a] MR spectra ofthe native corrinoids. 1,351.52 U 14 14 m/z 67 8 + , milli-absorbance units.( Dsf ∆ values (asshown by red arrows) confirmed thatthe =14.9 chromatogram and( 1 1,360 10 IC cobamide. Collectively,cobamide. ) revealed that the only notable changes (i.e., Dsf 2

Desulfovibriovulgaris ) ) 4 | ADV 16 16 1,366.48 N 3 b AL atoms inthelower basestructure. ( and cobamide. The superimposed superimposed The cobamide. D. hafniense B p T A 12 -cresol- he letters A, NCE ONLINE PUBLIC ( BI 3 d 1,380 7 B 18 4 18 ). indicate themaximumabsorbance za- ), 11.23 min; ), 11.23 OLO T Dsf ( he predominant corrinoid peakat C a f d b C ) H ba ( corrinoid (blackline)andthe 10 Absorbance (mAU) Absorbance (mAU) Vitamin B strain Y51 eluted at11.79 min ba ( Dsf 0 0 4 4 6 6 2 2 Dsf C C PLC 30 d cobamide 30 G B Vitamin Vitamin 12 9 obamide standards (5mg/ ) 03 12 0 B Cbi B 7 B ), 13.89 min;vitamin 1 B ( corrinoid. in Mass shifts H Y UV 12 b 12 andthe11.79-min native 12 and ), 17.23 minand17.64 min; chromatograms ofthe 86 12 A 55 ) upeetr Fg 5 Fig. Supplementary ( N B 36

D UV - 361 za- MR signals. 16 V 1 2 O corrinoid A Wavelength (nm) Wavelength (nm) 2 40 C is spectrumofthe 0 ; however,; hypoxan 400 TION | - 15 Dsf I C Dsf Ds V inred correlate the ) and the purified purified the and ) : 0

N isotope labeling isotope N ba ( f 1 is spectra +DMB p.p.m. 0 cobamide is 16 is cobamide . 1 5 0 www.nature.com/naturechemicalbiology Dsf 50 ), 13.34 min; ), 13.34 500 3 42 M 8 0 M cobamide. / + m/z N e 1 D f H NMR H C ) ) Mass M B H T 600 12 value 00 B he E

M L B 0

I - - - - ) O . 2 5 have any adjacent protons. Hydrogen atoms A, B, and C in not does aromaticmoiety the that suggesting molecule, the in tons the aromaticin protons base lower The region. aromatic the in differed spectra dechlorination activity ( of thecorrinoid recovered from dehalogenation enzymeassays. ( dechlorination activity ofdifferent of Dsf PC Figure 3|Identificationofpurinyl-Cba asthenative prosthetic group in by from able heteroatoms,amountextract purinyl-CbalimitedN of butthe the of each at bondingcovalent throughregioisomers four as exist can native of base lower assignmentunsubstitutedof the support the experimentsas purine moiety.These DMB the of p.p.m.) (2.2 group methyl adjacent the and p.p.m.) 6.5 and (7.3 protons benzyl the between correlations In purine. of atoms B hydrogenvitamin the comparison, three the with correlate directly 1 a c b 2 Dsf 13 (kDa) ladder Protein

C and/or C Signal intensity Signal intensity crudeprotein extracts usingBN–P 480 720 242 146 E 2 3 2 3 extraction 66 Corrinoid

× × × × R 10 10 10 10 10 10 Dase following nondenaturing,gel-electrophoretic separation 0 0 4 4 4 4 4 4 BN–PAGE gel Dsf

15 utrs i nt lo seeioe confirmation stereoisomer allow not did cultures Dsf N NMR spectroscopic analyses. Prior research has research Prior analyses. spectroscopic NMR N spectrum analysis Mass cobamide lacked a correlation with other pro other with correlation a lacked cobamide Relative Relative 46 46 Dsf Slice b abundance (%)

abundance (%) # 4 # 6 # 2 # 5 # 3 # 1 ) andthepurinyl- 100 100 50 50 0 0 cobamide. Purine-containing compoundsPurine-containing cobamide. 1,329.54 1,329.54 12 1,330 1,330 BN COSY spectrum demonstrated direct demonstrated spectrum COSY b BN , – Enzyme c assay Retention time(min) Retention time(min) P ) AG – U 1,340 1,340 P m/z m/z H AG E PLC gel slice #4showing thehighest A C E G ba standard (0.25mg/ gel slices measured using E separation andMSanalysis 1,351.52 1,351.52 1,350 1,350 . slice ( Gel # 4 # 6 # 2 # 5 # 3 # 1 a ) 0 TCE 0.5 28 0.5 0.2 0.5 [nmoles pervial] c ARTICLE -to- DCE produced 150 c DCE Purinyl-Cba Slice #4 8 8 354

Figure 2 300 L ) ( c ). 3 - - f

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. ARTICLE 4 spectrometry (UHPLC–HRMS) revealed that the corrinoid associatedultra-high-performance liquid chromatography–high-resolutionrecovered mass corrinoid from gel slice #4, but c not from slices #3 and #5, negligible (<8%) detectableand or exhibited slices no gel five other the rinationactivity wasfound associatedtobe with gelslice #4,whereas ( extracts crude JH1 strain proteins 242–480kDa region, presumably because of complexation with other nativePAGE blue(BN–PAGE)thefromexcised bands inwith gels associated generally is dechlorination activity reductivemaximum Mature Purinyl-Cba istheprosthetic group of ofaid corrinoids. discovery inthe new can andcharacterization base lowerapproach for practical a offers in other biomolecules. This combined suite of analytical techniques the precedence for purines to form bonds with an imidazole nitrogen stability: 9H > 7H > 3H > 1H) aromaticityofincreased because purineoverand 1H 3H (tautomer favoredare purine tautomersof purine 7H and 9H the that shown observed in of therespective lower basesubstrates. C of lower baseasthesubstrates. base for cobamide biosynthesis (indicated by the dashed line–enclosedpinkarea). ( substrate specificities. from phylogenetically diverse corrinoid-auxotrophic andprototrophic bacteria. Solidblackdotsindicate of respective specificity substrate and proteins homologous CobT of analysis Phylogenetic | 4 Figure DCE production( DCETCE from ob

A -R T N assays with -glycosidic bond was inferred to be analogous to that observed P [purine]shown inthepanelabove, butspectral analysis revealed distinctabsorbance features. ( 2 Dsf 5 Floig h nneauig eaain of separation nondenaturing the Following . b a A Dhc

-ribazole-5

PceA RDases are in the 50–60 kDa mass range enterica

Purine Desulfovibrio Salmonella

DMB

sticklandii

Clostridium Adenine type

vulgaris

C Desulfosporosinus reuteri Lactobacillus ob D CobT (purine)

M Desulﬁtobacterium T cochlearium Clostridium CobT assays withpurineasthelower base.( B C

` orpurineprovided asalower base substrate.

-phosphate ( ob multivorans Sulfurospirillum

T Dehalobacter NaMN enzymesofothermembers ofthe

Supplementary Fig. 6 Fig. Supplementary

100 100 T

100 parvula Veillonella CobT

he small peak to the left ofnicotiniche smallpeakto theleft acidinbottom trace ( 41

Nicotinic 33

Desulﬁtobacterium 100 55 67 100 31

acid

2 metallireducens Fig. 3 Fig. 68

3 22

. Based on these observations and on 31 13

100 62 A

limosum 100

100

-R

Eubacterium 0.8 thermoacetica Moorella 99 83 Dehalococcoides mccartyi

D Geobacter sulfurreducen Geobacter

100 Acetobacterium 65 Veillonella parvula P woodii a ata are averages ofmeasurements from duplicate assays. ) forms. ). Subsequent). in-gelextraction 91 Pelosinus fermentans Pelosinus fermentans

Geobacter lovleyi Geobacter Veillonella parvula Sporomusa ovata

propionicus

ArsB Pelobacter

Sporomusa ovata Dsf N -RP [purine]

-RP [DMB]

Phenol type Phenol s

), the highest dechlo highest the ),

Pce CobT ArsA N Bza type , nicotinic acidmononucleotide.( A e

NATURE ) MSanalysis of R . hafniense D. P Dases eptococcaceae may share catalytic features withthe 14, T 1 CH 6 he but , E de c A MIC NA 2 -RP [M] A262 (mAU) -R 4 - , 200 300 300 300 300 100 100

150 150 150 150 50 P A 0 0 0 0 0 0 concentrations were calculated on the basis of the concentration decreases A L T

-R other available with identity acid amino 58.5% than more no shares CobT whose for except Dsf type of cobamide produced ( forms syltransferase activates(CobT) lower tobases their respective phosphoribo nicotinate-nucleotide-dimethylbenzimidazole The Dsf nativegroup. prosthetic the as purinyl-Cba uses corrinoid detected in this gel slice, confirming that the no such proteins were detected in gel 5-methyltetrahydrofolate-homocysteine slice ple,#4. Purinyl-Cba methyltransferase), butwas the only exam Wood–Ljungdahl(forcorrinoid-dependent, enzymes pathway ( fumaratecorrinoid-independentreductase,botharewhich enzymes dinucleotide adenine (FAD)-dependent dehydro flavin monoxide and (CODH) genase carbon with along #4 slice gel in enriched was demonstratedand#5 #4, that the and times retention with gel slice #4 and the purinyl-Cba standard ( BIOLOGY upeetr Tbe 1 Table Supplementary URE NaMN NaMN NaMN NaMN NaMN P -RP [Purine] DMB c [ genomes are (81.3–100% highly conserved amino acid identity) CobT substrate specificity andphylogenetic analysis ) H D Dsf Dhc 24 M PLC Fig. 4 Fig. CobT

B CobT | ADV C Nicotinic Nicotinic Nicotinic b ] and ) HEM analysis demonstrating purified acid acid acid Dhc Dsf P euftbceim metallireducensDesulfitobacterium hylogenetic relationship of 40 40 of relationship hylogenetic A a Nicotinic NCE ONLINE PUBLIC

CobT. A C ), and the CobT substrate specificity determines the the determines specificity substrate CobT the and ), Dsf acid Purine -R ob Purine T IC P T Purine he (*)indicates thatno CobT sequences. A broader phylogenetic analysis [purine]formed in assay with purine) has a retention time similar to that m/z ( AL C d a Retention time(min) ob ) ) * values. The proteomic analysis of gel slices #3, T T T Dhc Dhc he he formation of Dsf Dsf ) BI enzymes with biochemically determined 26, with purine with purine with DMB with DMB Standards CobTassay CobTassay 1 CobTassay CobTassay Dsf 2 9 7 OLO . CobT sequences encoded in sequenced . CODH may occur in complexesinwith occur may CODH . Relative abundance (%) 68

A C TION | 100 100 ob 50 50 Dsf 0 0 Dsf 250 T G activated

PceA RDase (WP_011460641.1) C C www.nature.com/naturechemicalbiology Y ob ob A C A

C T D T -R ob -R 300 ob and activate purine as a lower enzymeassays. O -RP [DMB] -RP [DMB] P -RP [DMB] P T I T [ : [purine] production was

activity with 1 and homologous proteins m/z D 333.06 D Fig. Fig. 3b 0 M M . 1 tan S 15288, DSM strain 0 B B 35 DMB DMB -RP [purine] DMB 359.10 3 or purine to their ] or 04 8 / , c Dsf N A ) had matching C -R 10 H PceA RDase N P 00 E aM [purine] in M B N I O and a A . 2 -RP 5 1 2 - - -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. NA NATURE CH vitamin B addition ( whereas growth was negligible in control cultures without corrinoid B vitamin and purinyl-Cba- in 50-fold about ( corrinoid nous (5.4 8.2 amounts(TCE; trichloroethene ofsmall 23.7 ( day,respectively per statistically indifferent rates of 427 amount initial 64.4 ofthe B min vita of nM 36.9 received that culturesPER-K23 Strain Y51. strain of PceA the to identity acid amino 98.7% with RDase and ing webacteria, grew axenic organohalide-respir corrinoid-auxotrophic in dechlorination tive reduc corrinoid-dependent supports purinyl-Cba whether Totest E native prosthetic group of the as purinyl-Cba of discovery the with consistent purine, tuted demonstratedunprecedentedan and [DMB] 359.10 and 333.06 matching the calculated molecular mass of enzyme CobT containing fractions assay products showed strong adducts with on performed analysis MS of unsubstituted purine to base, or CobT, corroborating that No Dhc and only small amounts of nicotinate were observed, indicating that CobT,notpurinewasconsumed, ( period incubation 30-min a after replaced DMB, the conversion yield to ( period [DMB],respectively, were produced followingincubation 30-min a (250 DMBvated The RDase. PceA the of group guided DMB [DMB]; and that verified substrates as DMB or purine and (NaMN) ( DMB for vitro preference In a with bases lower benzimidazole-type family ( the to activation purine that suggested Peptococcaceae the of members these among tities strainsiden sequence have CobT identified.Thehigh not yet been either in producedcorrinoids native 56.6–61.7% amino acid identity, respectively ( tis meridiei youngiae Dehalobacter Dsf type lower base activation. The most closely related sequences to the phenol– in cated impli CobT other any from identity) acid amino (<52% distinct Table 2 duced by the respective host organisms ( pro cobamides native the about clues providedCobT, and terized characofactivated) type lower(i.e.,base substrate specificities the reflecting clades distinct into sequences CobT bacterial clustered upeetr Fg 7a Fig. Supplementary ffects ofpurinyl-Cbaffects ondechlorinatingactivity tan S 75 C030.) eoe, ih 785.% and 57.8–59.3% with genomes, CP003108.1) 765, DSM strain utvto wr idcts that indicates work Cultivation CobT clade(except CobT for A CobT was unable to activate unsubstituted purine ( purineunsubstituted activate to unable was CobT Dhc T Dhc o o -RP formation occurred in controls lacking NaMN, a lower a NaMN, lacking controls in occurred formation -RP 2.5 12 URE 9.3 ). This analysis revealed further that the (positivecontrol) purinyl-Cbaorcompletely dechlorinated Fig. 4 enzyme assays with purified purified with assays enzyme strain DSM 13257, CP003629.1; 13257, DSM strain CobT both activated DMB to the cultures with purinyl-Cba. Strain PER-K23 harbors a PceA i. 4 Fig. i. 4 Fig. tan S 174 CM001441.1; 17734, DSM strain E 12 Supplementary Table 3 MIC M M -fed mol), occurred in control incubations without exoge without incubations control in occurred mol), M Cl M A ( C b A Dsf R [uie, epciey ( respectively [purine], -RP d Dhb ). c L BIOLOGY HEM Dhb , osset ih h osrain ht exogenous that observation the with consistent ), . In ). Fig. 5 Fig. p to produce cobalamin as a non-native prostheticnon-native a as cobalamin produce to M − -cresol, adenine, guanine–hypoxanthine or Bza- or guanine–hypoxanthine adenine, -cresol, M), and 166.9 and M), released per day, resulting in the formation of formation the in resulting day, per released and ) cultures exclusively contained purinyl-Cba and Dsf Fig. 5 Fig. IC a

| ADV ). Strain PER-K23 cell numbers increased numbers cell PER-K23 Strain ). A oT sas n hc prn (250 purine which in assays CobT AL Desulfosporosinus o wt nctnc cd mononucleotide acid nicotinic with ) Dsf -RP derivative is a shared feature of this this of feature shared a is derivative -RP A D.metallireducens 2.3 a -RP [purine] ( Dehalobacter restrictus A ). Much lower dechlorination rates of rates dechlorination lower Much ). PceA. NCE ONLINE PUBLIC BI M Dsf mol of PCE to PCE ofmol Dsf OLO o ). Biomass from purinyl-Cba- and Dsf A o 2.70 and 159.4 and 2.70 -RP [purine] was not[purine]wasproduced, -RP 25 and 471 Dhc and i. 4c Fig. CobT specificity for unsubstifor specificity CobT CobT catalyzed the activation Dhb A G Fig. 4 oT tlzs vrey of variety a utilizes CobT Dsf -RP [purine] exceeded 99% A or Dhc Y Supplementary Fig. 7b Fig. 4 Fig. Desulfosporosinusorien -RP form of DMB ( (e.g., oT and CobT , m/z d

o D Desulfosporosinus b . n sas with assays In ). CobT) were CobT) found in CobT enzymes acti enzymes CobT Fig. 4 2.5 O A 12 and o TION | ([M+H] I Dsf -amended cultures, -amended c : 39 e

DCE within 6 d atd 6 within DCE 1 Desulfosporosinus Desulfosporosinus . hs findings These ). 0 M o CobT clade was strain PER-K23 Supplementary b . mol) andmol) M 1 1.13 0 www.nature.com/naturechemicalbiology ); however, the M Cl 3 D. hafnienseD. 8 + Dhc / Fig. 4c Fig. ) values of Dsf N M − released M C CobT CobT H CobT CobT c A A A E DCE DCE spp. M M Dhc -RP -RP -RP 10, , M) d B 1 ). ). 1 I ------. O

. 2 5 B of dechlorination reductive the with identity acid amino 20% VcrAthan andrespectively, lessBvcA share RDase, the which purinyl-Cba. to response ferent lamin as prosthetic the group. PceA RDases were fully functional with either purinyl-Cba or coba both that modifications did not occur ( B vitamin from three independentcultures. chloride ( filled diamonds, C amended with36.9 nMvitamin ( the onlycobamide recovered from purinyl- from vitamin ( 36.9 nMpurinyl- PCE ( activity ofcorrinoid-auxotrophic, organohalide-respiring bacteria. Figure 5| 1 c b a ba, ornocorrinoid addition(negative control). 12 2 ) Reductive dehalogenation of ) Reductive dechlorinationactivity in ) H b a -amended Corrinoid-auxotrophic astheelectron acceptor and36.9 nMvitamin A361 (mAU) PCE, TCE and cDCE (µmoles/bottle) PLC –0.4 40 40 40 60 60 60 20 20 20 0.0 0.4 0 0 0 0 2 1 10 chromatograms ofthetotal intracellular corrinoids extracted 00 VC E D. restrictusD. ffects ofpurinyl-Cbaffects substitution for vitaminB 12 ); filledcircles, ethene. , respectively, indicating that cobamide or lower base base lower or cobamide that indicating respectively, , B

12 24 Dhc - orpurinyl- TCE C ba, ornocorrinoid addition(negative control). tan A1 n sri G clue a rts of rates at cultures GT strain and BAV1 strain Purinyl-Cba ; filledtriangles, No corrinoid Purinyl-Cba Vitamin B 12 strain PER-K23 and PER-K23 strain 68 C ba-amended Dhc 12 B c c Fig. 5 Retention time(min) Dsf 12 DCE C t ehn ocre i vitamin in occurred ethene to DCE (positive control), 36.9 nMpurinyl- ue utrs eosrtd dif a demonstrated cultures pure E Time (d) Dhc and c rror bars represent mean values DCE to ethene in 14 b D. restrictus c ). These findings demonstrated strains BAV1 and GT express GT BAV1strainsand cDCE, VC and ethene (µmoles/bottle) ; inverted unfilledtriangles,vinyl Dhb C Dhb Vitamin B 40 40 40 80 80 80 60 60 60 20 20 20 ba-amended 0 0 0 cultures. PceA RDases. Complete RDases. PceA U D. hafniense D. nfilled squares, 12 cultures suppliedwith B Dhc 12 (positive control), 10 16 strain ARTICLE P Purinyl-Cba Dhb urinyl- No corrinoid Vitamin B 12 20 onthe cultures. B A strain Y51 strain C V

PCE ba was 1 cultures 12 30

18 o ; s.d.

5 - -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. ARTICLE a pronounced preference for DMB over unsubstituted purine as the the RDases, PceA the to contrast ( purinyl-Cba received thatcultures in robust enumerationindicatedactivity,dechlorinationrRNAgene tive 16S and rinoid carryover with the inoculum), and formed no ethene ( initial the of respectively,7.3%, and 22% than more no dechlorinated GT strain BAV1and strain corrinoid, exogenous without cultures control In supplied initially the of After a 32-d incubation period, the cultures dechlorinated about half ( day,respectively per released slower corrinoidamendment sole purinyl-Cbathe withas exhibited much and the hoststhe are unclear. In parasite (bilharzia) miasis plants and cultures cell in effects various against antibiotic yokosukanensis con that compound fungus biological the from isolated known first was Nebularine purine. unsubstituted only tains the is (purine nucleoside) nebularine date, To itself. purine to assigned been never has function biological specific a andrare, are moiety purine tuted FADH and (NAD otide 5 adenosine as such molecules ubiquitousand acids, nucleic of blocks building purine derivatives include adenine and guanine, which are essential systems biological in functions essential many fulfill derivatives purine and nature, in heterocycle taining nitrogen-con distributed widely most the is structure purine The DISC ity of corrinoid-dependent RDases lower base, emphasizing that the lower base can modulate the activ 6 enzyme corrinoid-dependent of modulators are structures base lower that emerges concept From a reports, systems. recent from biological and study in this purine unsubstituted to function a assigns and structures base lower functional occurring, naturally of number the expandsPeptococcaceae, the organohalide-of membersrespiring in purinyl-Cba molecules, of family corrinoid the biosynthesis corrinoid for machinery the have Archaea and Bacteria of members some onlybut life, branchesof all organismsin forfunctions bolic tate detailed investigations of its therapeutic potentials. therapies. synthesisPurinyl-Cbadisease using new enabling potentially signaling), (purinergic functions cellular of regulation the affect could nucleoside) purine (or purine tuted bases free corresponding the releases common hydrolasesnucleosidases) nucleosidepurine (i.e., the of hydrolysis enzymatic demonstrated and been has nebularine to adenosine of conversion enzymatic simple the established, not is mammals in purine tuted progression disease affect can that functions crucial fulfill they wherein tissues, mammalian adenosine-responsive the purinoceptors, P1 are all found almost in health implications. Purinergic membrane receptors, which include human potential has that finding a xenobiotic, a not is purine that and Eukaryota synthesize unsubstitutedBacteria purine, indicating enzymesingle a by adenosine of deamination reductive the of catalysis involves 201

orni-eedn ezm sses ufl esnil meta essential fulfill systems enzyme Corrinoid-dependent o Supplementary Fig. 8 Fig. Supplementary Supplementary Fig. 8 Fig. Supplementary 11 and 197 U c Dhc SSI Clitocybe nebularis Clitocybe DCE DCE dechlorination rates of 17.6 growth in cultures amended with vitamin B vitamin with amended cultures in growth 2 O ) + ` triphosphate (ATP), nicotinamide adenine dinucle 2 and NADH), and flavin adenine dinucleotide (FAD dinucleotide adenine flavin and NADH), and N c 8 3 Rmral, opud cnann a unsubsti an containing compounds Remarkably, . DCE to vinyl chloride (presumably enabled by cor by enabled (presumably chloride vinyl to DCE 5 3 . Apparently, . at somemembersdomainsof least the 0 and a a and o 10 M 1, Microbispora 2 M Cl , c 39– S. yokosukanensisS. 36– DCE (83 DCE Mycobacterium 2 4 3 9 2 ). Consistent with the observed reduc observed the with Consistent ). 3 . tan A1 n sri G cultures GT strain and BAV1 Strain ). 4 and is also produced by Te icvr o a e mme of member new a of discovery The . 8 ; however, its biological functions in in functions biological its however, ; − . Although the occurrence of unsubsti released per day, respectively ( 2 4 Fig. 5 Fig. Tu, h frain f unsubsti of formation the Thus, . 32, Dhc N 1 M 3 0 3 isolate . gyoii bn ctlzd by catalyzed bond -glycosidic , and is toxic to the schistoso the to toxic is and , mol) but produced no ethene. no produced but mol) c and RDases BvcA and VcrAhad and BvcA RDases Supplementary TableSupplementary 3 o species, exhibitsspecies, cytotoxic 1.2 and 11.7 2 , nebularine biosynthesis nebularine , 3 8 Supplementary Fig. 8 Fig. Supplementary 1 Ovos xmls of examples Obvious . . . Nebularine is a potent Dsf CobT will facili NATURE Streptomyces o 0.7 12 but not but de novo de M Fig. 5 Fig. 5 M Cl CH ). In ). E MIC NA 3 ). 5 c c ------− ,

A L T

16. 15. 14. 13. 12. 11. 10. M published online6November 2017 R diseases. human relevant of progression affect to options treatment expand to and example,(foranaerobicdigestion,production) calbiogas processes biotechnologi and bioremediation) example, (for environmental manipulateto avenues new provide may systems enzyme pendent corrinoid-dekey of base-controlledactivitylower of principle the and mammals,including organisms, of majority the for functions metabolic essential fulfill Cobamides membrane. cytoplasmic the periplasmicofrespiratoryenzymes suchas through exportRDases, case the in maturation,or,holoenzyme affect may structures base tionduring catalysis resolved,notconfigurabase-offisbut recenttion findingsa reveal extents) and manipulate, example, possibly (for activity and catalytic predict, to understood be must ments emphasizingtems, enzyme-specificthatcorrinoidcofactor require sys enzyme corrinoid-dependent other many to expand possibly function 9. 8. 7. 6. 5. 4. 3. 2. 1. R o the in available are references, and codes accession Methods, including statements of data availability and any associated BIOLOGY URE f the papef the eceived 14February 2017; accepted 2October 2017;

eferences

strain Y51. strain of dehalogenase reductive PceA the of characterization 16 chloroform. and 1,1,1-trichloroethane both dechlorinates genus. Biochem. bacterium homoacetogenic the from cobamide (2013). 1902–1911 incorporation of benzimidazole bases into cobamides. into bases benzimidazole of incorporation Environ.Microbiol. in metabolism corrinoid-dependent supports mccartyi organohalide-respiringin extents and rates dechlorination phylum the within order bioremediation, belong to a novel bacterial class, class, bacterial novel a to belong bioremediation, and cycling halogen to relevant organohalide-respiring bacteria anaerobic B for (2014). 455–458 Catal.ACS B the in dehalogenation of dehalogenase reductive tetrachloroethene Technol. Environ.Sci. 2,3,3,3-tetra refrigerantgeneration next the of corrinoid. Dehalospirillummultivorans (2011). acids. nucleic and proteins of ligands and 1999). Inc., Sons, & Wiley (John Environ.Microbiol. Suyama, A., Yamashita, M., Yoshino, S. & Furu Yamashita,Yoshino,& A., M., S. Suyama, A J.He, & S. Zhao, C., Ding, Villemur, & Eisinger,H.-J. E., Stupperich, Mo S. Yi, Yan,J. Lö Payne, M. Bommer, Liao, J.Maillard, J.-W.Walshe-Langford,Moon,Lö J., G.E., Im,& K Gruber, R T enz, P.enz, in räutler,B. , 3387–3397 (2014). 3387–3397 , f HO k er, F.E.er, , 12

R et al. et K | ADV Dehalococcoidales C -dependent dehalogenation. -dependent 10, et al. et FEMS Microbiol.FEMS .-Z., Chen, S.-L. & Siegbahn, P.E.M. Which oxidation state initiates stateP.E.M. oxidation WhichSiegbahn, & S.-L. Chen, .-Z., K .C. & Taga, M.E. Growth inhibition of inhibition Growth Taga,M.E. & .C. DS K . HEM 12

.A.P. 186 ISME J. ISME r R ., Pu ., , Helv.Acta Chim. Versatility in corrinoid salvaging and remodeling pathwaysremodeling and salvaging corrinoid Versatilityin . 20

Chemistry and Biochemistry of B12 of Biochemistryand Chemistry 4 A ., Lanthier, M., Beaudet, Beaudet, Lanthier,M., ., 5 J. Bacteriol. J. et al. et

et al. et et al. et 3 , T , 7350–7358 (2015). 7350–7358 , 2 NCE ONLINE PUBLIC . The exact role(s) of the lower base in RDase func RDase in base lower the of role(s) exact The . , 657–661 (1989). 657–661 , et al. et 1 et al. et f . This concept applies to the the to applies concept This . e corrinoid cofactor of reductive dehalogenases a dehalogenases reductive of cofactor corrinoid e er, B. & B.er, Characterization of the corrinoid iron-sulfur protein iron-sulfur corrinoid the of Characterization

Dehalococcoidesmccartyi T 10 Structural basis for organohalide respiration.organohalide for basis Structural

Chloro

78 69 R e cofactor of tetrachloroethene reductive dehalogenase of dehalogenase reductive tetrachloroethene of cofactor e , 1092–1101 (2016). 1092–1101 , 7, AL

eductive dehalogenase structure suggests a mechanism mechanism a suggests structure dehalogenase eductive 48 8 , 7745–7752 (2012). 7745–7752 , , 4628–4638 (2003). 4628–4638 , .If not directly involved in catalysis, the lower

ord. nov. and family 184 12 K , 13181–13187 (2014). 13181–13187 , R -dependent enzyme Np enzyme -dependent f räutler, B. Vitamin B Vitaminräutler,B. ev. BI exi , 3419–3425 (2002). 3419–3425 ,

is norpseudo-B is 86

30 Desul . OLO , 3698–3716 (2003). 3698–3716 , Int. J. Syst. Evol. Microbiol. Evol. Syst. J. Int. , 706–733 (2006). 706–733 , K A räutler, B. Identiräutler,B. TION | f Dhc Nature tobacterium R . & Lépine, F.Lépine, & . G eutv dclrnto rates dechlorination reductive www.nature.com/naturechemicalbiology Y Chem. Soc. Soc. Chem. gen. nov.,nov.,sp.obligatelygen.

517

12 D Dehalobacter restrictusDehalobacter Dehalococcoidaceae , a new type of a natural a of type new a , Dehalococcoidesmccartyi f O (ed. Banerjee, Banerjee, (ed. Sporomusa ovataSporomusa 12 k uoropropene (HFO-1234yf). (HFO-1234yf). uoropropene , 513–516 (2015). 513–516 , Sporomusa ovataSporomusa I -derivatives-enzyme cofactors -derivatives-enzyme sp. strain P strain sp. awa, f : R

Dehalococcoidia 1 f dhA: Co dhA: er, F.E.er,fateEnvironmental 0 Dhc cation of phenolyl of cation T . 1 K R 0 e J. Bacteriol. J. . Molecular . ev. 3 desul RDases, but may but RDases, Desul 8

Environ.Microbiol. / 63 40 Dehalococcoides II N , Co , R f online version online , 625–635 (2013). 625–635 , , 4346–4363 , R C reductively tobacterium f .) 558–572 .) H tobacterium by fam. nov.,fam. I f Science . , or Co or , E classis nov.,classis ects ects Eur.J.

M 195 . Appl. B I , . O

0 Appl. 346 ? . 2 sp.

, 1

2 ------

Pharmacol. Phytochemistry adenosine. fromhydroxylamine of release enzymic involves ribofuranoside) inhibitors. transport nucleoside with combination in analogues nucleoside purine Phytochemistry Chem. Biol. novelafrom 9- producing 7 products. (1999). 1961–1966 coliEscherichia Science cluster.[Ni-4Fe-5S] a reveals dehydrogenasemonoxide carbon a of structure involvement. quinone obviating chain transport electron respiratory dependent in complex metabolism. tautomers. purine of aromaticity on Microbiol. by formed hypoxanthylcobamide-corrinoids and multivorans in dehalogenase reductive tetrachloroethene non-functional B Lond. Dehalococcoidesmccartyi 20 speci substrate CobT and availability ligand lower both on Dehalococcoidesethenogenes bacterium Y51. strain in dehalogenase reductive tetrachloroethene R & E.G. Brown, el & E.G. Brown, riboside. purine of metabolism the of study A G.B. M.P.Brown, Gordon, & Cooper, Na nebularine. of structure the On B. Lüning, & N. Löfgren, R & G. Cecchini, T.M.,C., Iverson,Luna-Chavez, Dobbe K Mo Stasyu A., Weber, D.H., Guimarães, K Yang,J.,Lö Yan,Y. Im, J.,& Cro Nona R , 225 (1953). 225 , T ubli osemeyer, H. osemeyer, A. einhold, eller, S. eller, alevic, V.Burnstoc & alevic, K , 1265–1274 (2013). 1265–1274 , k f f ouni, M.H., Messier, N.J. & Cha, S. Treatment of schistosomiasis by Treatmentschistosomiasis ofS. Cha, Messier,& N.J.M.H., ouni, URE amura, G. Studies on antibiotic actinomycetes. III. on III. actinomycetes.antibiotic on Studies G. amura, att, B.A. & Ashihara, H. Purine and pyrimidine nucleotide synthesis and synthesis nucleotide pyrimidine and Purine H. Ashihara, & B.A. att, s, T.S., Seth, E.C., Hazra, A.B. & Taga, M.E. Cobamide structure depends depends structure Cobamide Taga,M.E. & A.B. Hazra, E.C., T.S., Seth, s, k k k , A. , k a, H. a,

293

, O.A., Szaty O.A., , , H., Svetlitchnyi,V.,Huber,H., Gremer,L., , E 368 R MIC et al. et

Chem. Biodivers. Chem. ., Horan, A.C., Gunnarsson, I., Patel, M. & Truumees, I. Nebularine Truumees, I. & M. Patel, I., Gunnarsson, A.C., Horan, ., 162 Desul et al. et Biochem. Pharmacol.Biochem. , 1281–1285 (2001). 1281–1285 ,

. Appl. Environ.Microbiol.Appl. R Dehalococcoidesmccartyi 220 , 20120320 (2013). 20120320 , et al. et C Environ.Microbiol. ArabidopsisBoo B A ev. et al. et Environ.Microbiol. Microbispora , 272–276 (1994). 272–276 , Exogenous 5,6-dimethylbenzimidazole caused production of a of production caused 5,6-dimethylbenzimidazole Exogenous - L BIOLOGY HEM

fumarate reductase respiratory complex.respiratory reductase fumarate D T K K 38 37 Identi

, 927–937 (1956). 927–937 , f -ribofuranosylpurine. 50 onu onu Complete genome sequence of the dehalorespiring the of sequence genome Complete tobacterium hafniense tobacterium e chemodiversity of purine as a constituent of natural ofconstituent a as purine of chemodiversity e , 61–71 (1995). 61–71 , (1994). 1589–1592 , Impact of vitamin B vitamin of Impact , 413–492 (1998). 413–492 , ł k k owicz, H. & & H. owicz, , M. Biosynthesis of nebularine (purine 9- (purine nebularine of Biosynthesis M. , riboside). (purine nebularine of cytotoxicity Plant M. , f k IC cation of a multi-protein reductive dehalogenase reductivemulti-protein a of cation , G. , reductive dechlorination activity.dechlorination reductive

| ADV sp.

AL 1 195. f R , 361–401 (2004). 361–401 , k K eceptors for purines and pyrimidines. and purines for eceptors

er, F.E. Guided cobalamin biosynthesis supports biosynthesis cobalamin F.E.er,Guided J. Ind. Microbiol. Ind. J. 1 laiber, I., Vogler,laiber,I., & B. A

, e0018 (2002). e0018 , 36

16 NCE ONLINE PUBLIC BI 18 J. Bacteriol. J. K , 3815–3821 (1987). 3815–3821 , , 3361–3369 (2014). 3361–3369 , , 3044–3056 (2016). 3044–3056 , rygows

strain CBDB1 suggests a protein- a suggests CBDB1 strain OLO 78 J. Org. Chem. Org. J. J. Antibiot.J. 12 Y51 and comparison with comparison and Y51 , 8025–8032 (2012). 8025–8032 , on formation of the the of formation on k i, T.M.E i, G Desul

188

Y 1 R R , 275–276 (1986). 275–276 ,

Desulfovibriovulgaris , 2262–2274 (2006). 2262–2274 , ees, D.C. Structure of the the of Structure D.C. ees, 14

. & Meyer, O. Crystal Crystal Meyer,O. & . f D

tobacterium hafniensetobacterium 77 , 94–97 (1961). 94–97 , O A R f TION | I , 4035–4045 (2012). 4035–4045 , enz, P.enz, Guanylcobamide ect of the H-bonding H-bonding the of ect :

1 Science 0 Acta Chem. Scand. Chem. Acta Streptomyces f . Phil. Trans.Phil. 1 city. 0 www.nature.com/naturechemicalbiology Sulfurospirillum 3 8

B 284 Chem. Biol. Chem. / - D N - , C H .

R Arch. E . Soc. Soc. . M

I O . 2 5 43. 42. 41. 40. 39. 38. 37. ported byported agrant from Superfund the Research Program under National the Institute Mass facility, Spectrometry Toronto, for LC/MS assistance. This research was sup ing We thank J. Maillard, École Polytechnique Fédérale deLausanne, France, for provid A to J.Y. or F.E.L. institutional affiliations. Correspondence and requests for materials should addressed be Nature remains neutral with regard to claims jurisdictional inpublished maps and is available at are available inthe Any supplementary information, chemical compound information and source data A The authors declare no competing interests. financial Competing financialinterests and interpretation, and J.Y., E.A.E.,and F.E.L. S.R.C., wrote manuscript. the analysis. N.J. generated analyses. P.W., O.M. and A.T.Q. BN–PAGE, performed enzyme assays, and proteomic tion, and phylogenetic analyses. A.K.B. and A.T.F. LC–MS performed and structural B.S., Y. Yang, and Y. Yin cultivation performed work, corrinoid and extraction purifica F.E.L., J.Y., conceptualized and S.R.C. research the and experiments. designed J.Y., M.B., A Scholarship and Council University the of Tennessee. supportfinancial from One-Hundred China-UT the Scholars Program by China the of Canada(NSERC) Industrial Biocatalysis Network to E.A.E.Y. Yin acknowledges the project ER-2312) to F.E.L. and by Natural the and Science Engineering Research Council provided by Strategic the Environmental Research and Development Program (SERDP of Environmental Health (R01ES024294)to F.E.L., Sciences with additional support 1 2 cknowledgments dditional Information uthor contributions

Dehalobacter restrictus gut microbial ecology.microbial gut 1987). Co., & Gruyter MedicineEcology, (2010). 3228–3259 vitamins. complex most the and compounds metalorganic 339 (2011). t halorespiration and between parallels reveals reductase (oQ) epoxyqueuosine growth. tumor system. Degnan, P.H., Taga, M.E. & Goodman, A.L. Vitamin B Vitamin A.L. Goodman, P.H.,& Degnan, Taga,M.E. in Z. Schneider, R Par McCarty, Z.D., Miles, AdinolVirgilio,F.& Di Burnstoc andaccio, L., Geremia, S., Demitri, N. & Wuerges,B VitaminJ.& N. Demitri, S., Geremia, L., andaccio, k , 1332–1335 (2013). 1332–1335 , s, J.M. s, R NA modi NA Nat. htt k , G. Purinergic signalling and disorders of the central nervous nervous central the of disorders and signalling Purinergic G. , p://www.nature.com/reprints/index.htm et al. et

R online vers ev. Drug Discov. Drug ev. Oncogene

Comprehensive B12: Chemistry,Biochemistry,ComprehensiveNutrition,B12: f T (eds. Schneider, Z. & Stroi & Z. Schneider, (eds. cobT cation. e genetic basis for bacterial mercury methylation. mercury bacterial for basis genetic e strain PER-K23. We Flick from thank R. BioZone the also expression clones. authors All contributed to data analysis Cell Metab.Cell R f ion of pape the .M., Molnar, G. & Bandarian, V. Discovery of of V.Discovery Bandarian, & G. Molnar, .M.,

, E. Extracellular purines, purinergic receptors and receptors purinergic purines, Extracellular E. , 36 Proc. Natl. Acad. Sci. USA Sci. Acad.Natl. Proc. , 293–303 (2017). 293–303 ,

7 , 575–590 (2008). 575–590 ,

20 , 769–778 (2014). 769–778 , r . Reprints and permissions information ń s k i, A.) 93–104 (Walterde 93–104 A.) i, l . Publisher’s note: Springer

108 12 ARTICLE as a modulator of modulator a as , 7368–7372 7368–7372 , Molecules 12 : unique :

15 Science

- , - -

7 © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. NATURE evaporator at 45 °C for residues2 h. The rotary dry were suspended in 75 a using dried vacuum and tubes plastic 2-mL new to transferred solutionwas the before corrinoids, solubilize to h 2 for °C tubes 60 closed at incubated The were extraction. corrinoid for 5.5) pH (final KCN mM 10 and acid acetic mM 50 containingmethanol (v/v) 90% of mL 1 with tubes plastic ratedbyBN–PAGE, andweretransferredgelslices excised individual 2-mL to (WatersCorp) cartridge Sep-Pak C18 a using purified and protocol extraction KCN established an following biomass the from extracted were corrinoids intracellular The Sciences). Life 0.22- diameter 47-mm 15,000 at centrifugation by sions extraction. Corrinoid tion process) ( unlabeled N in the upper CN ligand, which was added during the KCN extrac shift of 12.96 Da, close to the valueexpected of 13 total N atoms (14 minus one sulfurreducens of spectra mass the that Wefound structure. base lower the in atoms N of number the mine form) cyano the in atoms N (14 5-OHBza-Cba produces natively that bacterium a LC–MS analysis. Initial experiments with sufficient extract to medium of L 1.8 containing sel ves 2.2-L a to up scaling before medium of mL containing100 bottles serum experiments. labeling the 15 in omitted was and source nitrogen potential a is beled NH the dark at 30 °C. For methanol as the sole substrate sole substrate Sporomusa acids amino required of source as g/L) (0.5 as peptone acceptor,and mM) electron (0.48 PCE neat donor, electron an as hydrogen mL 10 source, bon described D.restrictus as acceptor electron as mM) (0.48 PCE neat and mM) (10 vate Sulfurospirillum described multivoransas acceptor electron an as fumarate mM 10 and donor electron sulfurreducens Geobacter B vitamin example, (for cobamide a and acceptor, electron donor,neat amended with 5 mM acetate as a carbon source, 10 mL hydrogen as an electron centration) as electron acceptor (68 PCE mentablesubstratewith and fer as mM) (10 pyruvate with supplemented were cultures PCE1 and Viet1 headspace. v/v) (80/20; eral salts medium, the Wolin vitamin mix excluding vitamin B 100 mL of bicarbonate (30 mM) or phosphate (50 mM) buffered, defined min Cultures. other wereAll chemicals used reagent or grade higher. ( mol 28,060 of coefficient extinction molar a using UV-Vis35 Lambda a with (PerkinElmer)spectrometernm at361 determined A and barkeri and Cba (5-OHBza-Cba), norpseudo vitamin B biosynthesis cobamide guided via prepared were 5-OMeBza-Cba purchased from Becton, Dickinson and Company. Bza-Cba, 5-MeBza-Cba and obtained from Cambridge Isotope Laboratories. Yeast Sigma-Aldrich. extract and 98%), peptone from were purchased were (Bza; (98%) purine and benzimidazole (97%), 98%), (5-OMeBza) (5-MeBza; 5-methoxybenzimidazole 5-methylbenzimidazole (99%), (DMB) ylbenzimidazole Chemicals. O q NH Y 99) n vnl hoie ( chloride vinyl and 99.9%) N 7 8 Sporomusa L 4 0 4 Cl-growncultures were 160-mL in transferredtwice v/v) inoculum; (1% IN 7 5 strain Fusaro (DSM 804), (DSM Fusaro strain , demonstrated, utility ofthe the 5 CH 9 E Pure cultures were grown in 160-mL glass serum bottles containingbottles serum glass 160-mL in grownwereculturesPure 4 p Cl Cl as the nitrogen source. . sp. strain KB-1 cultures were amended with betaine (50 mM) as the 1

E Ceo-b) ee xrce ad uiid from purified and extracted were -Cresol-Cba) Vitamin B ME strainPER-K23 cultures acetatewerecarmM as 5 amended with c cls rsetvl. ocnrtos f uiid oaie were cobamides purified of Concentrations respectively. cells, ) MIC DCE (79 DCE 2 0 cultures amended with amended cultures Supplementary Fig. 9 . sp. strain KB-1 (16S rRNA gene GenBank accession number accession GenBank gene rRNA (16S KB-1 strain sp. Methanosarcina barkeri A T 15 L HO -aee vru ulbld -Hz-b poue in produced 5-OHBza-Cba unlabeled versus N-labeled

BIOLOGY 15 12 M el wr hretd rm .–. L utr suspen culture L 0.3–1.8 from harvested were Cells N isotope experiments, DS M ( mol per bottle, 0.60 mM aqueousmM bottle,concentration)0.60 mol per anas D. hafniense D. m pore size polyethersulfone membrane filters (Pall filters membrane polyethersulfone size pore m q tan C clue rcie 5 M ctt a an as acetate mM 5 received cultures PCA strain 98%), dicyanocobinamide (Cbi; strain DSM 12446 cultures were grown with pyru 2 0 q . All vessels were incubated without agitation in 4 4 95) ee ucae fo Sigma-Aldrich. from purchased were 99.5%) . 1 Dhc × 0 . To extract corrinoids from proteins sepa proteins from corrinoids To extract . L Sulfurospirillummultivorans

-cysteine, a reductant added to the medium, g ). strains Y51 and JH1 and JH1 and Y51 strains for 15 min at 4 °C, or by filtration onto filtration by or °C, 4 at min 15 for strain BAV1 and strain GT cultures were M 12 15 strain Fusaro was grown with 123 mM mol per bottle, 0.48 mM aqueous conaqueous mM 0.48 bottle, per mol 15 and phenolic cobamides (i.e., Phenol- NH N isotopeN labelingapproach todeter Geobacter sulfurreducens 4 Cl versus NH versus Cl 15 NH 4 Cl Cl (0.3 g/L) replaced unla − 15 1 N-labeled corrinoid for corrinoid N-labeled cm 4 15 q − Cl returned a mass a returned Cl 1 NH 93%), 5,6-dimeth (ref. 12 ) as described as ) 12 Methanosarcina , and a N (DSM 12446) (DSM 4 Dsf l 9% was (99%) Cl 1 strain PCA, 1 0 M sp. strains sp. . Factor III Factor . 2 L ice-cold . Ethene ). 2 /CO G. 4 2 4 1 6 1 5 0 ------2 . . . .

(positive control) and individual gel slices inside an anoxic chamber (Coy (Coy chamber anoxic an inside slices gel individual and control) (positive were with activity conducted dechlorinating by the protein visualized standards in lane. assays the Enzyme ladder to detect as ranges mass molecular on based cut were slices gel The protocol Fisher). (Thermo staining G-250 Coomassie fast the to according stained and rated sepa were lanes sample and ladder blank, V. The 200 at min 45 another for then and V 150 at min 60 for chamber occurred BN–PAGE separation and bath, a ice an in and placed buffers chilled using done was Electrophoresis trometerFisher)(Thermo and UV-Visa described as nm to361 detector set spec mass Orbitrap Plus Exactive high-resolution a with tandem in system fromBN–PAGE extracted UltiMategelsanwereusinganalyzed UHPLC 3000 tinct from the 361 nm absorption ofpeak vitamin B dis nm, 355 at peak absorption an with UV-Visspectrum an exhibited and entire the lacks Cbi ligand. upper the study carried the cobalt atom in the fully oxidized +3 state and a cyano group as regionUV the in ancespectrum absorb corrinoid the influence coordination base lower and state, oxidation cobalt ligand, upper detector.The array diode a using collected were nm 600 to 250 between spectra UV-Visconditions. column initial to restored being before min 5 for B 75% at held period, time 3-min additional an over 75% to further and 12-min a over 25% to linearly increased B eluent of fraction the before B, eluent 18% and A eluent 82% was composition phase mobile initial The B. eluent as methanol in acid formic (v/v) 0.1% and A eluent as water in using 0.1% (v/v) formic acid ( diameter an with performed Eclipse was XDB-C18 column (Agilent Technologies, analysis 5 HPLC UPLC–HRMS. and spectroscopy, analysis. Corrinoid at 4°Cremoved insoluble Blue Coomassie dye and gel residuals. ammonium acetate (20 mM, pH 6.0), and centrifugation at 13,000 ing ing approximately 6 were with 25 loaded Protocol NativePAGEGel Bis–Tris the following performed was Blue Coomassie with stained Fisher) (Thermo Bis–Tris gel gradient 16% to 4 were prepared as described protein extracts crude analysis. proteomic hafniense and D. assays enzyme (BN–PAGE), PAGE native Blue scans was 1.5s,and total the measuring was time 1h. consisted of 256 scans per increment at 96 increments. The delay time between experiments NMR COSY gradient-selected The h. 1 of time measuring total a and selection PRESATsuppression solventusingpeak multiple scans, 1,024 The (Varian).system spectroscopy NMR methanol- in (250 dissolved was corrinoid purified of mg 0.7 Approximately nm. 361 atresponse detector the byinformed outlet detector the from lection obtainedfollowingseparationmanualwascontainingHPLC and col fraction predominantcorrinoid- the and extracted, Totalwere corrinoids intracellular TCE amendments (a total of 1,200 (0.5 (400 TCE yielded approximately 22 L of cell culture suspension. Each vessel received neat spectroscopy.NMR corrinoid. of ng 2.5 was method LC–MS this for limit detection The 140,000. of lution source (Thermo Fisher) with a ionization electrospray II HESI a using mode ionization positive in collected before equilibration to initial column conditions. Corrinoid weremass spectra hold 0.3-min a with min 5.56 after B eluent 90% and min, 3.32 after B eluent 50% min, 0.81 after eluentB 15% to increases linearfollowed by min, 0.46 for B eluent 0% and A eluent 100% was phase mobile The B. eluent as methanol min 1.9 Fisher; (Thermo column C18 Gold Hypersil a on separated were Corrinoids M − M m pore size, 2.1 mm inner diameter M 1 at 30 °C using 2.5 mM ammonium acetate in water as eluent A and 100% M) to enhance corrinoid production. Cells were harvested after three after harvested were Cells production. corrinoid enhance to M) L) to conduct × M 250 mm length) operated at a flow rate of 1 mL per min at 30 °C 30 at min per mL 1 of rate flow a at operated length) mm 250 L; 2.3 mM aqueous concentration), yeast extract (2 g/L) and Cbi and g/L) (2 extract yeast concentration), aqueous mM 2.3 L; strain JH1 cells were harvested from 100 mL cultures and the the and cultures mL 100 from harvested were cells JH1 strain 1 IntracellularUV-VisHPLC,bywere analyzedcorrrinoids Growth ofGrowth H and COSY NMR experiments using an INOVA 600 MHz M M L L of protein standards or crude protein contain extracts g of total protein as estimated with the Bradford assay the with g protein of as estimated total q 88%, w/v; 88%, Thermo w/v; Fisher, Waltham, MA, USA) m/z D. hafnienseD. 4 2 . All corrinoidsamples All and. standards this in M scan ranging from 1,000–1,500 and a reso L) were completely dechlorinated to A × -ribazole-5 50 mm length) at a flow rate of 0.2 mL 1 H NMR experiments consisted of consisted experiments NMR H strain Y51 in twelve 2.2-L vessels 2.2-L twelve in Y51 strain D. hafniense D. 4 ` 9 M doi:10.1038/nchembio.2512 -phosphate( 12 . . BN–PAGE pre-cast using m pore size, 4.6 mm inner ( Fig. 2 strain JH1 cell lysate b ). The corrinoids A × -RP) moiety -RP) 4 9

. Gel lanes lanes Gel . g for 5 min c DCE. DCE. d4 5 4 0 8 ------. .

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. doi:10.1038/nchembio.2512 dilutions of respective the plasmid DNA standards. preparedindependentlyweregeneratedserial three from standardandcurves cultures, triplicate from extracted samples DNA template used assays qPCR 4.6 with reaction, per The assay spanned a linear range from 4.64 R a 40.41, interceptof The insertion. a Technologies)with (Life pMK-RQ plasmid of dilution serial a using generated were curves Standard conditions. qPCR identical used probe and primers designed newly the with TaqMan and Dhc1240probeprobe Dhc1200F/Dhc1271R set primer using protocols established following described as BIO) (MO (Merck Millipore Ltd.) and processed using the MO BIO Soil DNA Isolation kit via vacuum filtration onto a 0.22 harvested were suspension culture mL 1 from cells DNA,genomic To obtain of the Cl to- dechlorination step is associated with the release of one chloride ion ter and end products, TCE/ and the PCE- c described as chromatograph gas 7890 Agilent an using cultures triplicate in quantified analyses. molecular and Chemical using BLAST search against NCBI the database. (5 (5 CGGACAAGCCGAGAAAATTC-3 (5 Df_pceA_1223F primer forward the of specificity The sequences. probe %, mol 80 the or primer the to either in bases C 30 or G consecutive three than more between no with was probe and primers the of content G+C The primer melting temperature ( a (ii) bp, 50–150 between size ampliconan (i) of criteria design (qPCR) PCR the of region consensus A Biosystems). (Applied software Express Primer using C other in identified tity) and DEHRE_12145), # (locus K23 in gene design. probe and Primer peptide unique two least at had identifications. that those include to filtered were which 99% than greater of probability Prophet Protein a at valid considered were Peptide a at 95% valid than greater of probability Prophet as accepted were identifications Peptide rate. false discovery a establish to database decoy reverse a with Inc.) Software, (Proteome 4.5.3 version software Scaffold using performed was refinement and dation vali Further modifications. peptide possible as Tandem X! in allowed were acid pyroglutamic or pyroglutamine to acid glutamic or glutamine of zation cycli or loss, ammonia N-terminal tryptophan, and methionine of oxidation glutamine, and asparagine of Deamidation Da. 2.5 of tolerance ion parent a and Da 0.40 of tolerance mass ion fragment a using proteins) 33,950 of (total database, as well as common contaminants such as human keratins and trypsin other RDases, characterized all genome, Y51 strain the on based peptides tryptic for search to up set was Tandem GPM, (The identification peptide/protein h for Tandem X! using Canada) Toronto,Toronto,of (University Facility Spectrometry Mass BioZone the at using gas chromatography was incubated in an anoxic chamber for 24 h and analyzed for mixture assay Each TCE. mM 2 and viologen, methyl mM 2 citrate, titanium 2 mM with amended Tris–HClmM 7.4) 100 (pH containing buffer vials glass described as Laboratory) DCE-to-ethene ( DCE-to-ethene t A t ` ` − -6FAM-TACGCGAGTTCTGCCG-MGB-3 -GCATCCGCACATTTTTTGC-3 p c released per day. The list of potential molecular formulas for the lower base D DCE DCE and Dsf : / 2 / Dsf w 8 and 7 w D. hafniense D. 9 cobamide was generated using platform web-based the ChemCalc 1 w 2 0 . The two-step dechlorination processes PCE-to- processes dechlorination two-step The . . . 1 t Dhb pceA Dhb h , c DCE-to-ethene DCE-to-ethene dechlorination rates were reported as micromolar e C g pceA p D m Dhc X . 0 PR sa sadr cre a a lp of slope a had curve standard assay qPCR o strain Y51 (locus # DSY2839), # (locus Y51 strain 2 r ) were measured based on the quantification of daugh of quantification the on based measured were ) pceA 2 9 g gene sequences was selected to meet the quantitative the meet to selected was sequences gene of 0.999, and a PCR amplification efficiency of 83.8%. of amplificationefficiency PCR a and 0.999, of 7 4 / 5 ; version X! TandemVengeance(2015.12.15.2)) X! version ; 4 . 4 Dsf 9 . Dhc 4 1 . The assays (1 mL) were conducted in 2-mL sealed sealed 2-mL in were conducted mL) (1 assays The . gene copies per reaction as the detection limit. All All limit. detection the as reaction per copiesgene 9 c D. hafniense D. Primers and TaqMan probe targeting the the targeting probe TaqMan and Primers , . . In-gel and digestion MS analysis were performed DCE andDCE vinyl chloride/ethene, respectively. Each and T A 5 m 16S rRNA gene-targeted qPCR was performed performed was qPCR gene-targetedrRNA 16S 3 A ) of 58–60 °C, and (iii) a probe . The qPCR assay targeting the the targetingassay qPCR The . M O m polyvinylidene difluoride membrane filter Dhb 6 Ethene and chlorinated compounds were compounds chlorinated and Ethene 0 ` ), the reverse primer Df_pceA_1286R Df_pceA_1286R primer reverse the ), ` 1 ad h poe Df_pceA_1247probe probe the and ) pceA 0 tan (eBn acsin numbers accession (GenBank strains 1 strain Y51 strain 5 . 1 1 . Subsequent protein identifications identifications protein Subsequent . ooos >5 sqec iden sequence (>95% homologs and × 10 ` ) to ) 1 to 4.64 A I pceA A pceA Dsf 5 D. restrictus D. 8 6 genes was verified by verified was genes × proteins in the NCBI NCBI the in proteins 8 fragment (1,656 bp) (1,656 fragment 10 0 . 1 8 c

) were designed designed were ) pceA c T DCE ( DCE DCE DCE formation m Dhb pceADhb of 68–70 °C D. hafniense D. strain PER- strain − gene copies .8, y- a 3.785, Dhb) gene 4 pceA and 9 . X! X! . 5 5 5 ` 2 4 3 - - - - - , . .

eaA102) ee mlfe uig hso Fah ihFdlt PCR High-Fidelity Flash Phusion using amplified were DehaBAV1_0626) tag. hexa-histidine express and clone to used was Millipore) (EMD CobT. of purification and expression Heterologous with 100bootstraps and and substitution Le the Gascuel model constructed was proteinsusingPHMYLmaximum the treebuilder plug-in likelihood Geneious in 8.1.7 homologous and CobT of tree phylogenetic The iterations. 10 with 8.1.7 Geneious in plug-in MUSCLE the using aligned and ( bacteria other from sequences ArsB) homologousand andArsAprotein (i.e., CobT 20 with along genomesofnine biosynthesis DMB anaerobic sequenced a ( Omega Clustal with performed were comparisons identity analyses. phylogenetic and Bioinformatics provided inMicrosoft Excel 2016. two-sample The compared. pendent analysis. Statistical column (5 column XDB-C18 Eclipse an with performed was Separation nm. 262 to set detector array diode a with equipped system HPLC series Technologies1200 Agilent respective their and 13,000 at centrifugation by removed was protein precipitatedNaOH, M 5 with pH the neutralizing Following min. 1 for bath 15 of tion incubated at 30 °C for 30 min before the reactions were terminated by the addi Tris–HCl7.5 pH werebuffer.mM 50 vials assayThein DMBor purine either ~2.6 of concentration each reaction (300 vitro In experiments. all for retained in liquid nitrogen and stored at solutions ( by was SDS–PAGEexamined The protein stock Blue staining. and Coomassie assay Bradford the using estimated were concentrations Protein glycerol. (w/v) 5% and imidazole mM 250 containing buffer HEPES of mL 5–7 with eluted were proteins imidazole), mM (20 buffer HEPES with washing about 0.5 mL After Ni-NTAcontaining column (Qiagen). glass resin centrifuged was lysate The off). 38,000 4-s at and on (3-s min 45 for bath ice an in (50 300 mM HEPES, NaCl pH and and 7.5), sonicated 10 mM imidazole, collected by centrifugation at 9,000 were r.p.m. Cells 180 and °C 16 at overnight incubated and Fisher) (Thermo 0.8. Cultures were induced with 0.4 mM isopropyl (OD nm 600 at density optical r.p.m. an to 180 and °C 37 at kanamycin 50 with Fisher) (Thermo Broth Terrific of L 1 in grown was plasmid modifications England BioLabs) for overexpression and purification. DehaBAV1_0626) (carrying ( pNJ049 and DSY2114) (carrying pNJ050 tors recombinationgous pre-induced with cells and vector prepared The constructs. supercoiled remaining remove to extracted bonedigestedandwas EcoRI NdeI,with dephosphorylated rSAP,with and gel back vector pET-28a(+) The BIO). (MO Kit Clean-Up PCR UltraClean the and Y51 ( NJ461-NJ462 and NJ459-NJ460 sets primer and Fisher) (Thermo MasterMix Supplementary Table 5 Supplementary Table 4 TableSupplementary c . u A Enzyme expression and purification were performed as described with with described as performed were purification and expression Enzyme k cobT Life Sciences Reporting Summary / T o CobT activity. CobT o Dhb Dhc inserts were then co-transformed into BW25113 electrocompetent BW25113 into co-transformed then were inserts l × M Dsf s M Dsf /

L formic acid ( acid formic L m g m pore size; 4.6 mm inner diameter inner mm 4.6 size; pore m cultures grown withpurinyl-Cba or vitamin B strain BAV1 axenic cultures. PCR products were cleaned using cleaned were products PCR cultures. BAV1axenic strain for 20 min and the supernatant was passed through a 25-mL 25-mL a through passed was supernatant the and min 20 for Y51 CobT, 15 mg/mL; mg/mL; 15 CobT, Y51 5 s genomes and search for orthologous for search and genomes 8 a . In brief, . In brief, / Dsf c l M u The PCE-to- The L) contained 30 5 s strains,five s cobT Dsf A 7 t Supplementary Table2 Supplementary . Following sequence verification, the recombinant vec recombinant the verification, sequence Following . a -RPs were analyzed by injecting 10 injecting by analyzed were -RPs M l L o oT sas ee efre a described as performed were assays CobT M), 2 mM NaMN, 10 mM MgCl mM 10 NaMN, mM 2 M), Red recombinase from plasmid pKD46 for homolo for pKD46 plasmid from recombinase Red ) were introduced into ) ), respectively, using genomic DNA from DNA genomic usingrespectively, ), E. coli 5 5 q . Reciprocal BLAST analysis was used to query the the query to used was analysis BLAST Reciprocal . 88%; w/v) and then transferred to a boiling water boiling a to transferred then and w/v) 88%; t 4 -test was carried out using the data analysis tool analysis data the using out carried was -test 7 lcs DY14 and DSY2114) # (locus . Twenty CobT amino acid sequences from the the from sequences acid amino Twenty CobT . − strain BL21(DE3) carrying a a carrying BL21(DE3) strain 80 80 °C. tag The hexa-histidine was N-terminal Dhb c DCE dechlorination rates from three inde three from rates dechlorination DCE × M

Dhc strainsand six g g CobT (corresponding to a final enzyme for 20 min, suspended in HEPES buffer BAV1 CobT, 10 mg/mL) were frozen for this paper is available. is paper this for RDase sequence alignments and alignments sequence RDase ) were retrieved from GenBank, GenBank, from retrieved were ) E. coli cobT B × × NATURE CH - 250 mm length) at a flow a at length) mm 250 D

g Desulfosporosinus -1-thiogalactopyranoside The pET-28a(+) vector pET-28a(+) The for 5 min. Lower bases bases Lower min. 5 for strain BL21(DE3) (New carrying an N-terminal N-terminal an carrying bza 5 genes implicated in implicated genes 0 h cobT Dhc and protein purity purity protein and M 2 12 , and 0.25 mM of mM 0.25 and , L samples into an into samples L were statistically E cobT h MIC 5 6 t . t p A expression expression : / L BIOLOGY lcs # (locus / 5 Dsf 9 w Briefly, . strains, w M strain 600 w g/mL g/mL . e ) of ) b i - - - - - . © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. 46. NATURE 45. 44. publishedthis articleand its supplementary information files. availability.Data to 400 200 of range mass a with MS high-resolution via detected and mode positive in ionizationelectrosprayoperated using ionized wereanalytes The injection. A (10 Aliquots Fisher). (Thermo dryer vacuum ISS110 300 combined,concentratedto then andmL tenfold 3 from the and wereperformed injectionssuch Three detected. was detectoroutletfrommanuallytarget the once peak the collected to100 above with minor modification as follows. The injection volume was increased outlined method the using performed was HPLC semi-preparative analysis, B,eluent15% A, B, 85% eluent A; parametersand time forgradient elutionthe were follows:as phase mobile initial The B. eluent as methanol in acid formic (v/v) 0.1% and rate of 1 mL min per at 30 °C using 0.1% (v/v) formic acid in water as eluent A -RP solutions were introduced into an Exactive Plus Orbitrap MS via direct via MS OrbitrapPlus Exactive an into introduced weresolutions -RP

in an anaerobic respiration.anaerobic an in trichloroethene and tetra- dechlorinates reductively that bacterium anaerobic (2012). Dehalococcoidesmccartyi from transfer cobamide interspecies polychloroethanes. e of capable Y51 strain Holliger,C. Yan,J., A. Suyama, m/z M L, and a 1-mL fraction containing fraction and1-mL L, a CH 1 R E 0 . MIC italahti, et al. et et al. et A t t All data generated or analyzed in this study are included in included are study this in analyzed or generated data All L = 5 min, 25% eluent B, 75% eluent A; = 15 min, endTomin, of15 = run. obtain material for subsequent MS

BIOLOGY Isolation and characterization of characterization and Isolation

K Dehalobacter restrictusDehalobacter .M., Wagner,Lö .M., D.D.& Biosci. Biotechnol.Biochem.Biosci. f cient dehalogenation of tetrachloroethene and tetrachloroethene of dehalogenation cient strains. Arch. Microbiol.Arch. Appl. Environ.Microbiol.Appl. Geobacter gen. nov. and sp. nov., a strictly strictly nov.,a sp.nov. and gen. A f -RP [DMB]-RP or

169 er, F.E.speci er,Unexpected A spp. to organohalide-respiringto spp.

-RP fractions from each were each from fractions -RP 65 , 313–321 (1998). 313–321 , , 1474–1481 (2001). 1474–1481 , Desul M L) of the concentrated the of L) t = 11 min, 85% eluent f tobacterium

A 78 M -RP [purine]was-RP t L using a Savanta using L , 6630–6636 , = 0, 15% eluent15% 0, = sp. f city of city 56. 55. 54. 53. 52. 51. 50. 49. 48. 47. 59. 58. 57.

Bioinformatics so Omega. Clustal using alignments sequence infrastructure. Environ.Microbiol.Appl. multiplemonitorssimultaneously genes dehalogenase (2003). 4646–4658 spectrometry. mass tandem by proteins identifying search. database and identi peptide of accuracy the estimate to model Biochem.Anal. binding.protein-dye of principle the utilizing protein of quantities microgram (2013). 974–981 electrophoresis. gel polyacrylamide native blue 11 chloroform-respiringautotrophyin corrinoid USA Sci. Acad.Natl. Proc. lower axial ligand of vitamin (B vitamin of ligand axial lower speci damage-control.metabolite (2000). 6640–6645 in K F.Sievers, bloc building a ChemCalc: A. Borel, & Patiny,L. R Nesvizhs K ofquantitation the for method sensitive and rapid A M.M. Bradford, Tang,S. Wang,P.-H. A.B. Hazra, Hazra, A.B., Tran, J.L.A., CroTran,J.L.A.,A.B., Hazra, L. Huang, Datsen italahti, earse, M. earse, Nesvizhs A., eller, f , 626–640 (2017). 626–640 , Escherichia coliEscherichia ware platform for the organization and analysis of sequence data. sequence of analysis andorganization the for platform ware f city in CobT homologs reveals widespread preference for DMB, the the DMB, forpreference widespread reveals homologs CobT in city k o, et al. et k K ii, A.I., ii, et al. et et al. et K et al. et .M. et al. et .A. & Wanner, B.L. One-step inactivation of chromosomal genes chromosomal of Wanner,inactivation One-step& B.L. .A. et al. et Functional characterization of reductive dehalogenases by using by dehalogenases reductive of characterization Functional

J. Chem. Inf. Model.Inf.Chem. J. Fast, scalable generation of high-quality protein multipleprotein high-quality of generation scalable Fast, A family of metal-dependent phosphatases implicated in implicatedphosphatases metal-dependent of family A et al. et 72 Geneious Basic: an integrated and extendable des extendable and integrated an Basic: Geneious Anaerobic biosynthesis of the lower ligand of vitamin B vitamin of ligand lower the of biosynthesis Anaerobic , 1647–1649 (2012). 1647–1649 ,

, 248–254 (1976). 248–254 , R K eller, A., eller, k e -12 using PC using -12 Quantitative PC Quantitative ii, A.I., ii, f Anal. Chem. Anal. ned experimental annotation reveals conserved conserved reveals annotation experimental ned

112 , 2765–2774 (2006). 2765–2774 , Nat. Chem. Biol. Chem. Nat. K K f ol ol , 10792–10797 (2015). 10792–10797 , s, T.S. & Taga, M.E. Analysis of substrate of Analysis Taga,M.E. T.S.& s, k k 12 er, E. & Aebersold, & E. er, Aebersold, & E. er, ). R

products. Chem. Biol. Chem. 53 , 5383–5392 (2002). 5383–5392 , R , 1223–1228 (2013). 1223–1228 , targeting 16S r 16S targeting Mol. Syst. Biol.Syst.Mol.

12 Proc. Natl. Acad. Sci. USA Sci. Acad.Natl. Proc. Dehalobacter

Appl. Environ.Microbiol.Appl. 20 , 621–627 (2016). 621–627 , f k doi:10.1038/nchembio.2512 cations made by MS/MS by made cations for tomorrow’sfor chemical , 1275–1285 (2013). 1275–1285 , Anal. Chem. Anal. R R . A statistical model for model statistical A . statistical Empirical . R Dehalococcoides NA and reductive and NA

isolates. 7 , 539 (2011). 539 ,

75 k , top ISME J. ISME strains.

97 79 12 , . ,

nature research | life sciences reporting summary

Corresponding author(s): Frank Loeffler and Jun Yan Initial submission Revised version Final submission Life Sciences Reporting Summary Nature Research wishes to improve the reproducibility of the work that we publish. This form is intended for publication with all accepted life science papers and provides structure for consistency and transparency in reporting. Every life science submission will use this form; some list items might not apply to an individual manuscript, but all fields must be completed for clarity. For further information on the points included in this form, see Reporting Life Sciences Research. For further information on Nature Research policies, including our data availability policy, see Authors & Referees and the Editorial Policy Checklist.

Experimental design 1. Sample size Describe how sample size was determined. Triplicate measurements with independent experiment verification. Information was provided in the Method section. 2. Data exclusions Describe any data exclusions. No data was excluded. 3. Replication Describe whether the experimental findings were The experimental findings were reliably reproduced. reliably reproduced. 4. Randomization Describe how samples/organisms/participants were Culture samples were randomly allocated into experimental groups. allocated into experimental groups. 5. Blinding Describe whether the investigators were blinded to The investigators were blinded to group allocation during data collection and/or group allocation during data collection and/or analysis. analysis. Note: all studies involving animals and/or human research participants must disclose whether blinding and randomization were used.

6. Statistical parameters For all figures and tables that use statistical methods, confirm that the following items are present in relevant figure legends (or in the Methods section if additional space is needed).

n/a Confirmed

The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement (animals, litters, cultures, etc.) A description of how samples were collected, noting whether measurements were taken from distinct samples or whether the same sample was measured repeatedly A statement indicating how many times each experiment was replicated The statistical test(s) used and whether they are one- or two-sided (note: only common tests should be described solely by name; more complex techniques should be described in the Methods section) A description of any assumptions or corrections, such as an adjustment for multiple comparisons The test results (e.g. P values) given as exact values whenever possible and with confidence intervals noted A clear description of statistics including central tendency (e.g. median, mean) and variation (e.g. standard deviation, interquartile range)

Clearly defined error bars June 2017

See the web collection on statistics for biologists for further resources and guidance.

1 Nature Chemical Biology: doi:10.1038/nchembio.2512 Software nature research | life sciences reporting summary Policy information about availability of computer code 7. Software Describe the software used to analyze the data in this The two-sample t-test was carried out using the data analysis tool provided in study. Microsoft Excel 2016.

For manuscripts utilizing custom algorithms or software that are central to the paper but not yet described in the published literature, software must be made available to editors and reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). Nature Methods guidance for providing algorithms and software for publication provides further information on this topic.

Materials and reagents Policy information about availability of materials 8. Materials availability Indicate whether there are restrictions on availability of Not applicable unique materials or if these materials are only available for distribution by a for-profit company. 9. Antibodies Describe the antibodies used and how they were validated Not applicable for use in the system under study (i.e. assay and species). 10. Eukaryotic cell lines a. State the source of each eukaryotic cell line used. Not applicable

b. Describe the method of cell line authentication used. Not applicable

c. Report whether the cell lines were tested for Not applicable mycoplasma contamination.

d. If any of the cell lines used are listed in the database Not applicable of commonly misidentified cell lines maintained by ICLAC, provide a scientific rationale for their use.

Animals and human research participants Policy information about studies involving animals; when reporting animal research, follow the ARRIVE guidelines 11. Description of research animals Provide details on animals and/or animal-derived No animal studies were conducted. materials used in the study.

Policy information about studies involving human research participants 12. Description of human research participants Describe the covariate-relevant population No human subjects were used in this study. characteristics of the human research participants. June 2017

2 Nature Chemical Biology: doi:10.1038/nchembio.2512