PDF SI Purinyl cobamide NCB manuscript 9 25 17 Jun...4 Supplementary Table 2. Information about CobT...

1

Supplementary Information

Purinyl-cobamide is a native prosthetic group of reductive dehalogenases

Authors and affiliations:

Jun Yan1,2,3,4,5*, Meng Bi1, Allen K. Bourdon6, Abigail T. Farmer6, Po-Hsiang Wang7,

Olivia Molenda7, Andrew Quaile7, Nannan Jiang3,4,5,8, Yi Yang3,9, Yongchao Yin1, Burcu

Şimşir3,9, Shawn R. Campagna6, Elizabeth A. Edwards7, and Frank E. Löffler1,3,4,5,8,9,10*

1Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37996,

USA; 2Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of

Applied Ecology, Chinese Academy of Sciences, Shenyang, Liaoning 110016,

P.R.China; 3Center for Environmental Biotechnology, University of Tennessee,

Knoxville, Tennessee 37996, USA; 4Biosciences Division, Oak Ridge National

Laboratory, Oak Ridge, Tennessee 37831, USA; 5Joint Institute for Biological Sciences

(JIBS), Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA;

6Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996, USA;

7Department of Chemical Engineering and Applied Chemistry, University of Toronto,

Toronto, Ontario, M5S 3E5, Canada; 8Bredesen Center for Interdisciplinary Research

and Graduate Education, University of Tennessee, Knoxville, Tennessee 37996, USA;

9Department of Civil and Environmental Engineering, University of Tennessee, Knoxville,

Tennessee 37996, USA; and 10Department of Biosystems Engineering & Soil Science,

University of Tennessee, Knoxville, Tennessee 37996, USA.

��Corresponding authors: [email protected] and [email protected]

Nature Chemical Biology: doi:10.1038/nchembio.2512

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Supplementary Results

Supplementary Tables 1-5

Supplementary Figures 1-9


3

Supplementary Table 1. Proteomic analysis of the Desulfitobacterium (Dsf) hafniense strain JH1 proteins associated

with different BN-PAGE slices.

Gel slice #

Protein ladder

range (kDa)a

No. of distinct

peptidesb

Total spectralcountsb

Protein description NCBI accession no.

1 ³ 720 - - - - 2 480-720 - - - -

3 346-480 5 11 Pyruvate ferredoxin oxidoreductase WP_019849101.1 5 18 Acetyl-CoA synthase WP_005812973.1

4 204-346 6 8 CO dehydrogenase WP_011459977.1 4 6 Tetrachloroethene dehalogenase WP_011460641.1 5 11 FAD-dependent fumarate reductase WP_005817128.1

5 112-204

2 6 Pyridoxamine 5’-phosphate oxidase WP_005815111.1 7 10 FAD-dependent fumarate reductase WP_005817128.1 3 4 Substrate-binding ABC transporter WP_005812007.1 2 2 Peptidoglycan-binding protein LysM WP_015943955.1

6 £ 112 - - - -

a BN-PAGE lanes were separated into slices based on the position of proteins in a MW ladder. The MW ranges were

estimated from a standard curve created by interpolating the log MW of individual ladder proteins (146, 242, 480, 720 and

1,048 KDa) versus the ratio (Rf) of migration distance of each ladder protein to the migration distance of the Coomassie

Blue dye front. Note that the actual MWs of Dsf proteins do not necessarily match the calculated MWs based on BN-

PAGE separation.

b In-gel peptide digestion and proteomic analysis were performed on gel slices # 3, 4, and 5, but not # 1, 2, and 6.


4

Supplementary Table 2. Information about CobT and homologous proteins used for phylogenetic tree construction (Fig. 4b

in main text). Indicated are the host organisms, the designations of the CobT homologous proteins, the experimentally

determined or inferred (based on the native types lower bases produced in the respective host organisms) substrate

specificity, the locus tag numbers of the corresponding genes, and the NCBI accession numbers of the proteins.

Organism CobT homologue

Substrate specificity Locus tag NCBI

accession no. Reference

Acetobacterium woodii DSM 1030

CobT Bza type Awo_c14620 AFA48245.1 1

Eubacterium limosum strain KIST612

CobT Bza type ELI_4217 ADO39159.1 1

Geobacter sulfurreducens strain PCA

CobT Bza type GSU3009 AAR36401.1 1

Geobacter lovleyi strain SZ

CobT Bza type Glov_3678 ACD97377.1 1

Pelobacter propionicus DSM 2379

CobT Bza type Ppro_2806 ABL00406.1 2

Moorella thermoacetica ATCC 39073

CobT Bza type Moth_1721 ABC20023.1 3

Dehalococcoides mccartyi strain 195

CobT Bza type DET0657 AAW40094.1 4

Clostridium sticklandii DSM 519

CobT Adenine type CLOST_0282 CBH20412.1 5

Sulfurospirillum multivorans DSM 12446

CobT Adenine type SMUL_1547 AHJ12807.1 6

Clostridium cochlearium DSM 1285

CobT Adenine type SAMN05216497_102226 SDK92407.1 7


5

Veillonella parvula DSM 2008

CobT Adenine type Vpar_1602 ACZ25278.1 4

Lactobacillus reuteri DSM 20016

CobT Adenine type Lreu_1695 ABQ83934.1 4

Salmonella enterica Strain LT2

CobT Adenine type STM2016 AAL20920.1 4

Pelosinus fermentans strain R7 ArsA Phenol type N.A WP_007950675.1 8

Sporomusa ovata DSM 2662

ArsA Phenol type N.A AEG78648.1 9

Veillonella parvula DSM 2008 ArsA Phenol type Vpar_1456 ACZ25133.1 4

Pelosinus fermentans strain R7

ArsB Phenol type N.A. WP_007950676.1 8

Sporomusa ovata DSM 2662

ArsB Phenol type N.A WP_021169525.1 9

Veillonella parvula DSM 2008

ArsB Phenol type Vpar_1457 ACZ25134.1 4

Desulfovibrio vulgaris strain Hildenborough

CobT Guanine type DVU_3279 AAS97749.1 10

Desulfitobacterium sp. strain PCE1

CobT Purine DESPCE1_RS0113770 WP_014794544.1 This study

Desulfitobacterium hafniense strain Y51 CobT Purine DSY_RS11275 WP_018211350.1 This study

Desulfitobacterium dichloroeliminans

strain LMG-P21439 CobT Unknown Desdi_2285 AGA69715.1 This study

Desulfitobacterium dehalogenans ATCC 51507 CobT Unknown Desde_2751 AFM01064.1 This study

Desulfitobacterium sp. strain PCP-1

CobT Unknown N.A. WP_005812799.1 This study

Desulfitobacterium hafniense strain PCS-S CobT Unknown DPCES_2346 CDX02233.1 This study


6

Desulfitobacterium hafniense strain DCB-2 CobT Unknown DHAF_RS16390 WP_018211350.1 This study

Desulfitobacterium hafniense strain TCP-A CobT Unknown DESHAF_RS0103255 WP_018211350.1 This study

Desulfitobacterium metallireducens DSM 15288 CobT Unknown DESME_08705 AHF07147.1 This study

Dehalobacter sp. strain CF

CobT Unknown DCF50_p694 AFV04701.1 This study

Dehalobacter sp. strain DCA

CobT Unknown DHBDCA_p636 AFV01665.1 This study

Dehalobacter restrictus strain PER-K23

CobT Unknown DEHRE_07530 AHF09955.1 This study

Dehalobacter sp. strain UNSWDHB


Dehalobacter sp. strain E1 CobT Unknown N.A. WP_019225400.1 This study

Desulfosporosinus orientis DSM 765

CobT Unknown Desor_1444 AET67101.1 This study

Desulfosporosinus meridiei DSM 13257

CobT Unknown Desmer_1443 AFQ43436.1 This study

Desulfosporosinus youngiae DSM 17734

CobT Unknown DesyoDRAFT_1368 EHQ88526.1 This study

Desulfosporosinus sp. strain BRH_c37

CobT Unknown APF81_21415 KUO78188.1 This study

Desulfosporosinus sp. strain I2


Desulfosporosinus sp. strain HMP52



7

Supplementary Table 3. Growth of corrinoid-auxotrophic organohalide-respiring

Dehalobacter restrictus (Dhb) and Dehalococcoides mccartyi (Dhc) pure cultures with

different cobamides.

Organism (e- acceptor) Cobamide

Dhc 16S rRNA or Dhb pceA gene copies per mL culture a

Average fold

increase Initial Final

Dhb (PCE)

Vitamin B12 7.52 ± 0.25 x 106 3.89 ± 0.49 x 108 51.7

Purinyl-Cba 7.52 ± 0.25 x 106 3.75 ± 0.47 x 108 49.9

None 7.52 ± 0.25 x 106 1.70 ± 0.85 x 107 2.3

Dhc strain BAV1 (cDCE)

Vitamin B12 4.63 ± 0.22 x 106 1.83 ± 0.09 x 108 39.5

Purinyl-Cba 4.63 ± 0.22 x 106 1.29 ± 0.15 x 107 2.8

None 4.63 ± 0.22 x 106 6.26 ± 1.62 x 106 1.4

Dhc strain GT (cDCE)

Vitamin B12 3.88 ± 0.70 x 106 1.68 ± 0.14 x 108 43.3

Purinyl-Cba 3.88 ± 0.70 x 106 2.00 ± 0.31 x 107 5.2

None 3.88 ± 0.70 x 106 4.62 ± 0.51 x 106 1.2

a Based on the available genome information, Dhc possess a single copy of the 16S

rRNA gene and Dhb possess a single copy of the pceA gene. Average and standard

deviation of gene copy numbers were calculated from qPCR measurements of triplicate

cultures.


8

Supplementary Table 4. Nucleotide sequences of the primers designed in this study for PCR amplification of the cobT

genes to construct overexpression plasmids. Primer set NJ459/NJ460 was used to amplify the Dsf cobT gene (locus #

DSY2114) from Dsf strain Y51 genomic DNA. Primer set NJ461/NJ462 was used to amplify the Dhc cobT gene (locus #

DehaBAV1_0626) from Dhc strain BAV1 genomic DNA. The homology regions for vector recombination are highlighted.

Primer Sequence (5’-3’)

NJ459 TCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATGTGGATGACTTCTTAAGACAGGAAAAC

NJ460 TGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGTTAGTTTTTACCGCTGACTCCTGC

NJ461 TCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGAACTATTAAACGCAACACTTGC

NJ462 TGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGCTAACTTTGCTTTTCGGAAACCC


9

Supplementary Table 5. Information about the plasmids used in this study for the overexpression of CobT enzymes.

Plasmid Description Source or reference

pET-28a(+) General E. coli expression vector with IPTG induction EMD Millipore

pKD46 Red swap plasmid carrying λ Red recombinase 11

pNJ050 pET-28a(+) with IPTG-inducible expression of Dsf cobT (locus # DSY2114) carrying an N-terminal 6X-HIS tag This study

pNJ049 pET-28a(+) with IPTG-inducible expression of Dhc cobT (locus # DehaBAV1_0626) carrying an N-terminal 6X-HIS tag This study

IPTG: isopropyl β-D-1-thiogalactopyranoside


10

Supplementary Figure 1. The native corrinoids produced by several PCE-

dechlorinating Desulfitobacterium (Dsf) strains. (a) HPLC chromatograms of the

corrinoids extracted from axenic cultures of Dsf hafniense strain JH1, Dsf sp. strain

Viet1, and Dsf sp. strain PCE1. (b) The HPLC chromatogram of a cobinamide standard

(5 mg/L), which was separated into the monocyano- (8.77 min) and dicyano- (15.63 min)

forms.

A36

1 (m

AU

) A

361

(mA

U)

Retention time (min)

a

b

Dsf hafniense strain JH1

Dsf sp. strain Viet1

Dsf sp. strain PCE1

Cobinamide (Cbi) 8.77 min 15.63 min


11

Supplementary Figure 2. The UV-Vis spectra (250-600 nm) of the two compounds

that eluted after 15.74 min and 15.94 min showed absorption peaks at 355 nm, distinct

from complete cobamides in the cyano form. These compounds likely represent

cobinamide precursors.

Fraction at 15.74 min Fraction at 15.94 min

Wavelength (nm)

Abs

orba

nce

(mA

U)

355 nm


12

Supplementary Figure 3. The mass spectra of cobamides produced by different Dsf

strains and authentic vitamin B12. (a) The mass spectrum of the predominant Dsf native

corrinoid produced by Dsf hafniense strain Y51. (b) The mass spectrum of authentic

vitamin B12. The mass spectra of the predominant corrinoid produced by (c) Dsf

hafniense strain JH1, (d) Dsf sp. strain Viet1, and (e) Dsf sp. strain PCE1. (f) The mass

spectrum of the non-native cobamide produced in Dsf hafniense strain Y51 cultures with

25 µM DMB amendment.

Rel

ativ

e ab

unda

nce

(%)

Rel

ativ

e ab

unda

nce

(%)

Rel

ativ

e ab

unda

nce

(%)

Dsf hafniense strain Y51

Vitamin B12

Dsf hafniense strain JH1

Dsf sp. strain Viet1

Dsf sp. strain PCE1

Strain Y51+DMB

m/z m/z

Rel

ativ

e ab

unda

nce

(%)

Rel

ativ

e ab

unda

nce

(%)

Rel

ativ

e ab

unda

nce

(%)

a

b

c

d

e

f

1329.54

1329.54

1329.54

1329.54 1355.58

1355.58

1351.52

1351.52

1351.52

1351.52 1377.56

1377.56


13

Supplementary Figure 4. Guided cobamide biosynthesis by Dsf hafniense strain Y51.

(a) HPLC chromatograms of a benzimidazolyl-cobamide (Bza-Cba) standard (5 mg/L)

and the non-native cobamide formed in strain Y51 cultures amended with 25 µM

benzimidazole (Bza). (b) UV-Vis spectra (250-600 nm) of the Bza-Cba standard and

the non-native cobamide synthesized in strain Y51 cultures with an adsorption

maximum of 361 nm.


14

Supplementary Figure 5. Results from homonuclear correlation spectroscopy (COSY)

experiments. Gradient-selected COSY nuclear magnetic resonance spectra of (a) the

Dsf corrinoid (0.7 mg in 250 µL methanol-d4 solution) and (b) a vitamin B12 standard

(0.7 mg in 250 µL methanol-d4 solution).


15

Supplementary Figure 6. Representative image of a blue native polyacrylamide gel

electrophoresis (BN-PAGE) gel used for Dsf hafniense strain JH1 crude protein

separation and subsequent in-gel analysis of PceA reductive dechlorination activity.

Protein ladder

Protein ladder

Dsf crude

protein

Dsf crude

protein

480

242

146 66

20

720

1048

480

242 146

66

20

720

1048

kDa kDa


16

Supplementary Figure 7. Heterologous expression of Dsf CobT and Dhc CobT in E.

coli and requirements for lower base activation. (a) SDS-PAGE image of Dsf CobT and

Dhc CobT carrying an N-terminal hexa-histidine tag purified from recombinant E. coli

cells. (b) HPLC chromatograms of CobT enzyme assay controls demonstrating that the

formation of alpha-ribazole-5’-phosphate requires nicotinic acid mononucleotide (NaMN),

a lower base, and CobT.

b

Dsf CobT + DMB (no NaMN)

Dsf CobT + NaMN (no lower base)

Dhc CobT + DMB (no NaMN)

Dhc CobT + NaMN (no lower base)

NaMN + DMB (no CobT)

Dsf CobT

Dhc CobT

Protein ladder KDa

11 17 20 25

35 48 63 75 100

a A

262

(mA

U)

Retention time (min)

39.4 KDa 37.5 KDa

DMB

NaMN Nicotinic acid

NaMN

NaMN

DMB

DMB


17

Supplementary Figure 8. Reductive dechlorination of cDCE to ethene in Dhc strain

GT cultures receiving vitamin B12 (36.9 nM, top panel), purinyl-Cba (36.9 nM, middle

panel), or no corrinoid addition (negative control, bottom panel). Solid triangles, cDCE;

open, inverted triangles, vinyl chloride; solid circles, ethene. Error bars represent the

mean values ± standard deviation from three independent cultures.

cDC

E, v

inyl

chl

orid

e an

d et

hene

(µm

oles

/bot

tle)

Time (day)

Vitamin B12

Purinyl-Cba

No corrinoid


18

Supplementary Figure 9. Demonstration of the utility of 15N-labeling to obtain lower

base compositional information. Mass spectra of (a) unlabeled and (b) 15N-labeled 5-

OHBza-Cba (i.e., factor III) produced by Geobacter sulfurreducens strain PCA. The

15N-labeled 5-OHBza-Cba was purified from Geobacter sulfurreducens cells grown with

15N-NH4Cl as the sole nitrogen source. Mass shifts of 12.95 Dalton in the m/z values of

[M+H]+ and [M+Na]+ base peaks matched the predicted mass increase based on the

total number of two N atoms in the Bza lower base structure.

a

b

Rel

ativ

e ab

unda

nce

(%)

m/z

1343.54

1356.51

1365.53

1378.48

Unlabeled 5-OHBza-Cba R

elat

ive

abun

danc

e (%

)

N

N

OH 15N-labeled 5-OHBza-Cba


19

References

1. Hazra, A.B. et al. Anaerobic biosynthesis of the lower ligand of vitamin B12. Proc. Natl. Acad. Sci. USA 112, 10792-10797 (2015).

2. Cheong, C.-G., Escalante-Semerena, J.C. & Rayment, I. Structural investigation of the biosynthesis of alternative lower ligands for cobamides by nicotinate mononucleotide: 5,6-dimethylbenzimidazole phosphoribosyltransferase from Salmonella enterica. J. Biol. Chem. 276, 37612-37620 (2001).

3. Stupperich, E., Eisinger, H.-J. & Schurr, S. Corrinoids in anaerobic bacteria. FEMS Microbiol. Lett. 87, 355-360 (1990).

4. Crofts, T.S., Seth, E.C., Hazra, A.B. & Taga, M.E. Cobamide structure depends on both lower ligand availability and CobT substrate specificity. Chem. Biol. 20, 1265-1274 (2013).

5. Renz, P. in Chemistry and biochemistry of B12 (ed. Banerjee, R.) 558-572 (John Wiley & Sons, Inc., 1999).

6. Kräutler, B. et al. The cofactor of tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans is norpseudo-B12, a new type of a natural corrinoid. Helv. Chim. Acta 86, 3698-3716 (2003).

7. Hoffmann, B. et al. Native corrinoids from Clostridium cochlearium are adeninylcobamides: spectroscopic analysis and identification of pseudovitamin B12 and factor A. J. Bacteriol. 182, 4773-4782 (2000).

8. Men, Y.J. et al. Sustainable growth of Dehalococcoides mccartyi 195 by corrinoid salvaging and remodeling in defined lactate-fermenting consortia. Appl. Environ. Microbiol. 80, 2133-2141 (2014).

9. Chan, C.H. & Escalante-Semerena, J.C. ArsAB, a novel enzyme from Sporomusa ovata activates phenolic bases for adenosylcobamide biosynthesis. Mol. Microbiol. 81, 952-967 (2011).

10. Guimarães, D.H., Weber, A., Klaiber, I., Vogler, B. & Renz, P. Guanylcobamide and hypoxanthylcobamide-corrinoids formed by Desulfovibrio vulgaris. Arch. Microbiol. 162, 272-276 (1994).

11. Datsenko, K.A. & Wanner, B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640-6645 (2000).


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