Carbon monoxide dehydrogenase: A novel target for the development of new anti-tubercular drugs

Carbon monoxide dehydrogenase:A novel target for the development of

new anti-tubercular drugs

Young Min KimDepartment of Biology, Yonsei University

Molecular Microbiology Laboratory, Yonsei University

From Basic to Application

CO


- An air pollutant

- A color- and odorless toxic gas : high affinity to metal-containing enzymes (e.g. hemoglobin, a-type cytochromes)

- Also acts as a neurotransmitter : the one produced endogenously during heme degradation

Production and sources - 12 - 14 x 108 tons per year - Major source : incomplete oxidation of fossil fuels

* Constant concentration in atmosphere : 0.03 - 0.9 ppm (50 - 100 ppm in urban district)

Sinks - Major sink (2/3) : Oxidation by hydroxyl radical in the upper atmosphere (troposphere) - Biological oxidation, especially microbial oxidation, plays a major role in the lower atmosphere (soil and water)


Carboxydobacteria

- A group of aerobic bacteria which are able to grow at the expense of carbon monoxide as the sole source of carbon and energy

- Facultatively chemolithoautotrophs


Carboxydobacteria

- A group of aerobic bacteria which are able to grow at the expense of carbon monoxide as the sole source of carbon and energy

- Facultatively chemolithotrophic bacteria

- Gram(-) carboxydobacteria Mycobacterial carboxydobacteria (Gram-positive) Gram(+) carboxydobacteria other than mycobacteria


Carboxydobacteria


Gram negative - Pseudomonas carboxydohydrogena - Hydrogenophaga pseudoflava Oligotropha carboxidovorans

Gram positive Mycobacterium sp. strain JC1 ( Acinetobacter sp. strain JC1)2002

Bases of CO utilization

CO dehydrogenase

CO-insensitive electron transport system

Calvin cycle * Mycobacterium tuberculosis: reductive TCA cycle (?)


CO dehydrogenases

Aerobic bacteria - carboxydobacteria - metals: Mo, Cu, Fe

Anaerobic bacteria - acetogenic bacteria, methanogenic bacteria, phototrophic bacteria, sulfate reducers - metals: Ni, Fe


CO dehydrogenases in carboxydobacteria

300-380 kD

3 nonidentical subunits (L2M2S2)

Molybdopterin cytosine dinucleotide:[2Fe-2S]:FAD (2:4:2)

Inducible by CO * Mycobacterium sp. strain JC1: constitutive & CO-inducible


FAD

FeS II

FeS I

MCD-Mo

O. Meyer’s group

O. carboxidovorans


CO + H2O CO-DH(MCD-HCOO- + H+/[Fe-S]/FAD) CO2

CO-DH(MCD/[Fe-S]/FADH2) CO-DH(MCD/[Fe-S]/FAD) 2H+ + 2e-

Role of CO dehydrogenase during CO oxiadtion


CO + H2O CO2 + 2H+ + 2e-



Fate of electrons during CO oxidation


CO + H2O CO2 + 2H+ + 2e-

Organic materials

NADH UQ/Cyt. b Cyt. c Cyt. a O2

CO UQ/Cyt. b Cyt. o O2

NADH

Electron transport system in carboxydobacteria


CO2 fixation in carboxydobacteria


Calvin cycle

Reductive TCA cycle (Mycoabcterium tuberculosis?)

Hydroxypropionate pathway

Acetyl-CoA pathway

CO + H2O CO2 + 2H+ + 2e-



Oxidation of CO by CO dehydrogenase


CO + H2O CO2 + 2H+ + 2e-

Cellular

materials

Energy

Mycobacteria and CO


Reclassification of CO-oxidizing isolate


Acinetobacter sp. strain JC1 (1985)

Mycobacterium sp. strain JC1 (2002)

Mycobacterial carboxydobacteria

++

+ + + +

M. phlei

M. flavescens

+ + +M. peregrinum

+ + +M. neoaurum

+ + +M. Smegmatis MC2

+ + +M. gastri

+ + +M. vaccae+ + +

- + +M. tuberculosis H37Ra

+ + +Mycobacterium sp. strain JC1

M. parafortuitum

Growth on CO

CO-DHactivity

RubisCOactivityBacteria


Park et al., J. Bacteriol. 185:142-147 (2003)

Activity staining and Immunoblotting of CO-DHs in mycobacteria

1 2 3 4 5 6 7 8 9 10

A

B


Identity of amino acid sequence of Mycobacterium sp. strain JC1 CO-DH to those of CO-DHs in other bacteria

Large Medium SmallBacteria

O. carboxidovorans

P. thermocarboxydovorans

H. pseudoflava

M. tuberculosis

55.7 38.2 57.7

57.7 36.9 57.4

57.8 40.4 57.0

87.7 71.2 81.7

Amino acid identity (%)


* No immunological relationship between CO-DHs in mycobacteria and Gram-negative bacteria

moaE cutR cutB1 cutC1 cutA1 orf7 orf8 orf9 orf10 orf11 orf12 orf13modC cutB2 cutC2 cutA2 orf17 orf18 orf19 orf20 orf21

Mycobacterim sp. strain JC1

M. tuberculosis H37Rv

0376c 0375c 0374c 0373c 0372c 0370c0371c 0369c rv0377 0368c

0383c 0382c 0381c 0380c 0379c 0377c0378c 0376cmb0384 0375c

M. bovis0414c 0413c 0412c 0411c 0410c 0408c0409c 0407cbcg0415 0406c

M. bovis BCG

M. marinum0654 0655 0656 0657 0658 06600659 0661mmar0653 0662

M. smegmatis

0743 0744 0745 0746 0747 07490748msmeg0742 0750

cutM cutS cutL

H. pseudoflava

orf1

cutB cutC cutA orf4

O. carboxidovorans


0477 0478 0479 0480 0481 04830482mjls0476 0484

Mycobacterium sp. JLS0499 0500 0501 0502 0503 05050504mkms0498 0506

Mycobacterium sp. KMS0488 0489 0490 0491 0492 04940493mmcs0487 0495

Mycobacterium sp. MCS

coxC coxM coxS coxL coxD coxE coxGcoxB coxHcoxF coxKcoxI

trd_A0566 0565 0564 0563

T. roseum coxC coxM coxS coxL coxD coxE coxF coxG

R. jostii 0523205231rha1_ro05229 05230 05233 0523505234 05236

1 kb


CO-DH genes and conserved genes up- and downstream of the CO-DH genes in mycobacteria

Park et al., Microbiology 156:999-1008 (2010)

What is the physiological role ofCO dehydrogenase in

pathogenic mycobacteria?



MW: 28 MW: 30

http://jb.asm.org/cgi/content/full/185/1/142/F1

http://upload.wikimedia.org/wikipedia/commons/7/78/Nitric-oxide-2D.png

http://upload.wikimedia.org/wikipedia/commons/1/14/Carbon-monoxide-2D-dimensions.png

NO


- An air pollutant

- An important signaling molecule in human body- Beneficial and detrimental

- Kills bacteria in macrophages

CO-DH and NO-DH activities in mycobacterial and a Gram-negative carboxydobacteriaa


Park et al., BBRC 362:449-453 (2007)

a Activity was determined with cell-free extracts prepared from cells grown at 37oC in SMB-CO.b Nanomoles of INT reduced per milligram of protein per minute.

NO source: sodium nitroprusside (SNP)

BacteriaSpecific activityb

SourceCO-DH NO-DH

Mycobacterium sp. strain JC1 DSM 3803 M. tuberculosis H37Ra ATCC 35835M. vaccae ATCC 15483O. carboxidovorans OM5 DSM 1227

6.09.9

17.518.1

1.52.03.10.0

1 21 2

Lane 1: Stained for CO-DH (CO) Lane 2: Stained for NO-DH (SNP)

CO-DH and NO-DH stained by activity


Purified CO-DH of Mycobacterium sp. strain JC1

- CO-DH activity : 139.3 nmol of INT reduced/mg protein/min - NO-DH activity : 32.6 nmol of INT reduced/mg protein/min

Purified CO-DH possesses NO-DH activity



NO + H2O NO2 + 2H+ + 2e-

Oxidation of NO by NO-DH activity of CO-DH

CO + H2O CO2 + 2H+ + 2e-

CO-DH and NO-DH activities in cells incubated in the presence and absence of SNPa

a Cells were grown in SMB-CO in the presence (+SNP) and absence (-SNP) of 5 mM SNP. Activity was determined with cell-free extracts prepared from cells harvested at the indicated time intervals after inoculation of the CO-grown cells into the appropriate medium.b Nanomoles of INT reduced per milligram of protein per minute.


Bacteria

Specific activityb

CO-DH NO-DH

Mycobacterium sp. strain JC1

O. carboxidovorans OM5

02040

5.7 8.5 0.0

17.6 7.6 7.8

Time after inoculation

(min)

1.51.41.5

0.00.00.0

1.52.10.0

0.00.00.0

-SNP +SNP –SNP +SNP

5.75.76.0

17.617.014.5

02040

Effect of SNP on the expression of CO-DH

Immunoblotting of CO-DH

Mycobacterium sp. strain JC1 SMB-glucose + SNP

0 20 40(min)

Panel A: Mycobacterium sp. JC1, CO, stained with CBBPanel B: Mycobacterium sp. JC1, CO, stained by activityPanel C: Mycobacterium sp. JC1, glucose, stained by activityPanel D: O. carboxidovorans OM5, CO, stained by activity Cells were harvested at the time of inoculation (lane 1) and at 20 min (lane 2) and 40 min (lane 3) after inoculation

-SNP +SNP

1 2 3 1 2 3

A

B

C

D


Mycobacterial carboxydobacteria

++

+ + + +

M. phlei

M. flavescens

+ + +M. peregrinum

+ + +M. neoaurum

+ + +M. Smegmatis MC2

+ + +M. gastri

+ + +M. vaccae+ + +

- + +M. tuberculosis H37Ra

+ + +Mycobacterium sp. strain JC1

M. parafortuitum

Growth on CO

CO-DHactivity

RubisCOactivityBacteria


Park et al., J. Bacteriol. 185:142-147 (2003)




0376c 0375c 0374c 0373c 0372c 0370c0371c 0369c rv0377 0368c

0383c 0382c 0381c 0380c 0379c 0377c0378c 0376cmb0384 0375c


M. bovis BCG

M. marinum0654 0655 0656 0657 0658 06600659 0661mmar0653 0662

M. smegmatis

0743 0744 0745 0746 0747 07490748msmeg0742 0750

cutM cutS cutL

H. pseudoflava

orf1

cutB cutC cutA orf4

O. carboxidovorans


0477 0478 0479 0480 0481 04830482mjls0476 0484



Mycobacterium sp. MCS

coxC coxM coxS coxL coxD coxE coxGcoxB coxHcoxF coxKcoxI

trd_A0566 0565 0564 0563

T. roseum coxC coxM coxS coxL coxD coxE coxF coxG

R. jostii 0523205231rha1_ro05229 05230 05233 0523505234 05236

1 kb


CO-DH genes and conserved genes up- and downstream of the CO-DH genes in mycobacteria

Park et al., Microbiology 156:999-1008 (2010)

Tuberculosis


- A common and often deadly disease caused in human mainly by Mycobacterium tuberculosis

- Systemic but usually attacks lung

• Infects over a third of the world’s population (~2 billions)

- one in 10 asymptomatic latent infections progresses to active TB (8~9 millions per year)

• Accounts for 2 million death annually (one every 15-20 seconds)

• Infects a fourth of AIDS patients - accounts for a third of AIDS patient’s death


M. tuberculosis

http://en.wikipedia.org/wiki/Image:Mycobacterium_tuberculosis.jpg


RNSROS

RNS

Strategies of mycobacteria to survive inside host macrophages

1. Prevention of phagosome-lysosome fusion

2. Inactivation of toxic reactive oxygen species by KatG etc.

3. Inactivation of toxic reactive nitrogen species by; - NO dioxygenase activity of Hmp and trHbN - Peroxinitritase activity of KatG and alkylhydroperoxide reductase AhpC


ROS

RNS




3. Inactivation of toxic reactive nitrogen species by; - NO dioxygenase activity of Hmp and trHbN - Peroxinitritase activity of KatG and alkylhydroperoxide reductase AhpC - NO-DH activity of CO-DH (?)


ROS

Effect of CO-DH on the survival of E. coli under NO stress


E. coli DH5α, 5 mM SNP, 50 mM Tris-HCl (pH 7.5), RT 50 g of Mycobacterium sp. JC1 purified CO-DH

Effect of NO on the survival of Mycobacterium sp. JC1 cutR- mutant under NO stress

Mycobacterium sp. strain JC1 wild type and cutR- mutant cells, 5 mM SNP, RT


cutR cutB cutC cutA212 2555 2984 7669

2 3244 3564 3906 4686 6916 7152

2 3167 3487 3829 4609 6839 7075

2464 2907 7592

BamHI

EcoRI

(base)

BamHI

EcoRI

(base)

Copy 1

Copy 2


2 3244 3564 3906 4686 6916 7152

2 3167 3487 3829 4609 6839 7075

2464 2907 7592

BamHI

EcoRI

(base)

BamHI

EcoRI

(base)

Copy 1

Copy 2

Effect of NO on the survival of Mycobacterium sp. strain JC1 cutA- mutant under NO stress



2 3244 3564 3906 4686 6916 7152

2 3167 3487 3829 4609 6839 7075

2464 2907 7592

BamHI

EcoRI

(base)

BamHI

EcoRI

(base)

Copy 1

Copy 2


2 3244 3564 3906 4686 6916 7152

2 3167 3487 3829 4609 6839 7075

2464 2907 7592

BamHI

EcoRI

(base)

BamHI

EcoRI

(base)

Copy 1

Copy 2

Effect of NO on the survival of M. smegmatis cutA- mutant under NO stress


cutR orf2cutC cutAcutB orf4 orf5 orf6orf1cutR orf2cutC cutAcutB orf4 orf5 orf6orf1

Survival of M. tuberculosis H37Rv in macrophage


Wild type-1Wild type-2CO-DH mutant#1-1CO-DH mutant#1-2CO-DH mutant#2-1CO-DH mutant#2-1

0372c0374c 0373c0375c 0371c 0370c0376c0377 0369c 0368c0372c0374c 0373c0375c 0371c 0370c0376c0377 0369c 0368c0372c0374c 0373c0375c 0371c 0370c0376c0377 0369c 0368c0372c0374c 0373c0375c 0371c 0370c0376c0377 0369c 0368c




3. Inactivation of toxic reactive nitrogen species by; - NO dioxygenase activity of Hmp and trHbN - Peroxinitritase activity of KatG and alkylhydroperoxide reductase AhpC

- NO-DH activity of CO-DH



Possible role of CO-DH in pathogenic mycobacteria

NOCO

CO-DH

Known anti-tuberculosis drugs and genes inducing resistance to the drugs

Anti-tuberculosis drugs Action mechanism

Genes involved in the resistanceMutation rate (%)Genes Products Roles

IsoniazidInhibition of mycolic acid biosynthesisInhibition of DNA, lipids, carbohydrates, and NAD metabolism

katG Catalase- peroxidase Prodrug conversion 20-80

inhA Enoyl ACP reductase Drug target 15-43

ndh NADH dehydrogenase II INH activity modulator 10

ahpC Alkyl hydroperoxidase Marker of resistance 10-15

Rifampin Inhibition of transcription rpoB RNA polymerase Drug target 96

PyrazinamideAcidification of cytoplasmLoss of membrane energy

pncA Nicotinamidase/pyrazinamidase Prodrug conversion 72-97

5-Chloro-pyrazinamide Inhibition of FASI fasI FASI Drug target NA

EthambutolInhibition of arabinogalactan biosynthesis

embCAB Arabinosyltransferases Drug target 47-65

Streptomycin Inhibition of protein synthesisrpsL S12 ribosomal protein Drug target 52-59

rrs 16S rRNA Drug target 8-21

Amikacin/kanamycin Inhibition of protein synthesis rrs 16S rRNA Drug target 76



Ethionamide Inhibition of mycolic acid biosynthesisetaA/ethA Flavin monooxygenase Prodrug conversion 37

inhA Enoyl ACP reductase Drug target 56


Known anti-tuberculosis drugs and genes inducing resistance to the drugs

Anti-tuberculosis drugs Action mechanism

Genes involved in the resistanceMutation rate (%)Genes Products Roles

IsoniazidInhibition of mycolic acid biosynthesisInhibition of DNA, lipids, carbohydrates, and NAD metabolism

katG Catalase- peroxidase Prodrug conversion 20-80

inhA Enoyl ACP reductase Drug target 15-43

ndh NADH dehydrogenase II INH activity modulator 10

ahpC Alkyl hydroperoxidase Marker of resistance 10-15

Rifampin Inhibition of transcription rpoB RNA polymerase Drug target 96

PyrazinamideAcidification of cytoplasmLoss of membrane energy

pncA Nicotinamidase/pyrazinamidase Prodrug conversion 72-97

5-Chloro-pyrazinamide Inhibition of FASI fasI FASI Drug target NA

EthambutolInhibition of arabinogalactan biosynthesis

embCAB Arabinosyltransferases Drug target 47-65



Amikacin/kanamycin Inhibition of protein synthesis rrs 16S rRNA Drug target 76



Ethionamide Inhibition of mycolic acid biosynthesisetaA/ethA Flavin monooxygenase Prodrug conversion 37

inhA Enoyl ACP reductase Drug target 56


Multi-drug resist

ant TB

Extensively drug-resistant TB

No new anti-TB drug has beenintroduced in the last four decades

→ needs novel targets with less mutation rate

CO-oxidizing system as a novel target for TB drug screening

1. CO dehydrogenase : screening for natural and synthetic compounds

2. Proteins involved in the expression of CO

dehydrogenase


0372c0374c 0373c0375c 0371c 0370c0376c0377 0369c 0368c0372c0374c 0373c0375c 0371c 0370c0376c0377 0369c 0368c


Functional analysis of genes (possibly) involved in the oxidation of CO in mycobactera


Mycobacterium smegmatis

Mycobacterium tuberculosis

Mycobacterium sp. strain JC1cutR orf2cutC cutAcutB orf3 orf4 orf5

orf2cutC cutAcutB orf3 orf4orf1 orf6

Copy I

Copy IIorf5

Genes Mutant Function Structure

cutR orf1cutBCAorf2orf3 orf4 orf5 orf6

regulation unknownCO-DH unknown Const. exp. (?) unknown unknownregulation

OXOOXOXO

OOXXOXXX

cutR orf2cutC cutAcutB orf4 orf5 orf6orf1

Ribbon diagram of CutR in Mycobacterium sp. strain JC1


cutR orf2cutC cutAcutB orf3 orf4 orf5

orf2cutC cutAcutB orf3 orf4orf1 orf6orf5

Ribbon diagram of Orf1 in M. smegmatis



Ribbon diagram of Orf3 in Mycobacterium sp. strain JC1


cutR orf2cutC cutAcutB orf3 orf4 orf5

orf2cutC cutAcutB orf3 orf4orf1 orf6orf5

Thank youfor

your attention




0376c 0375c 0374c 0373c 0372c 0370c0371c 0369c rv0377 0368c

0383c 0382c 0381c 0380c 0379c 0377c0378c 0376cmb0384 0375c


M. bovis BCG

M. marinum0654 0655 0656 0657 0658 06600659 0661mmar0653 0662

M. smegmatis

0743 0744 0745 0746 0747 07490748msmeg0742 0750

cutM cutS cutL

H. pseudoflava

orf1

cutB cutC cutA orf4

O. carboxidovorans


0477 0478 0479 0480 0481 04830482mjls0476 0484



Mycobacterium sp. MCScoxC coxM coxS coxL coxD coxE coxGcoxB coxHcoxF coxKcoxI

trd_A0566 0565 0564 0563

T. roseum coxC coxM coxS coxL coxD coxE coxF

R. pomeroyi

coxG

sace 0534 0537 0538 05390535 0536

S. erythraea

bxe_C0029 0030 0031 0032 0033 00350034rcoM

B. xenovorans chromosome 3

B. xenovorans chromosome 1

0036

bxe_A4360 4356 435443574358 43554359

04976 04975rha1_ro04979 0497704978

R. jostii 0523205231rha1_ro05229 05230 05233 0523505234 05236

Gra

m

(-)G

ram

(+

)m

ycob

acte

ria

1 kb

ABG06612

ABL89718

ABN96292

ABK70147

JC1

ACC39118

ABL02876

CAA17379

CAD93243

CAL70396

ABG97013

ACM06436

AAV95654

CAA57829

AAD00363

CAA54902

ABE30822

ABE35958

ABG96757

CAL99883100

100

99

100

98

100

63

74

100

100

91

100

100 83

81 43

69

0.10.1 substitutions per site

Gro

up

II

Gro

up

III

Gro

up

I

Thermomicrobium roseum Trd_A0564 (ACM06436)

Hydrogenophaga pseudoflava CutL (AAD00363)

Pseudomonas thermocarboxydovorans CutA (CAA54902)

Ruegeria pomeroyi CoxL (AAV95654)

Mycobacterium sp. strain JC1 CutA (FJ896490)

Oligotropha carboxidovorans CoxL (CAA57829)

Burkholderia xenovorans CoxL2 (ABE30822)

Burkholderia xenovorans CoxL (ABE35958)

Rhodococcus jostii RHA1_ro04976 (ABG96757)

Saccharopolyspora erythraea SACE_0537 (CAL99883)

Mycobacterium smegmatis MSMEG_0746 (ABK70147)

Mycobacterium sp. MCS Mmcs_0491 (ABG06612)

Mycobacterium sp. KMS Mkms_0502 (ABL89718)

Mycobacterium sp. JLS Mjls_0480 (ABN96292)

Mycobacterium marinum CoxL (ACC39118)

Mycobacterium ulcerans CoxL (ABL02876)

Mycobacterium tuberculosis Rv0373c (CAA17379)

Mycobacterium bovis Mb0380c (CAD93243)

Mycobacterium bovis BCG BCG_0411c (CAL70396)

Rhodococcus jostii RHA1_ro05232 (ABG97013)

CO-DH large subunit


Carbon monoxide dehydrogenase: A novel target for the development of new anti-tubercular drugs

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Transcript of Carbon monoxide dehydrogenase: A novel target for the development of new anti-tubercular drugs