Effect of molybdate and tungstate on the biosynthesis of CO dehydrogenase and the molybdopterin cytosine dinucleotide-type of molybdenum cofactor in Hydrogenophaga pseudoflava

Molecular microbiology Lab. Molecular microbiology Lab. 한승정한승정

AbstractAbstract The molybdenum-containing iron-sulfur flavoprotein CO dehydrogenase is express

ed during heterotrophic growth of the aerobic bacterium Hydrogenophaga pseudoflava with pyruvate plus CO.

Effect of molybdate and tungstate on the biosynthesis of CO dehydrogenase 1) Subunit structure 2) Cofactors 3) Mo-MCD cofactor : biosynthesis and its insertion

Purpose:Purpose:Tungstate inhibited chemolithoautotrophic growth of H. pseudoflava with CO, but had no effect on heterotrophic growth of the bacterium with pyruvate and thus provided the possibility to study the effect of molybdate and tungstate on the biosynthesis, structure and reactivity of CO dehydrogenase as well as the effect of the metals on the biosynthesis of the Mo-MCD cofactor and its insertion into the protein.

IntroductionIntroduction

The group VI elements tungsten (W) and molybdenum (Mo)

Mo and W are incorporated into proteins as the molybdenum cofactor (Moco), which contains a mononuclear Mo or W atom coordinated by one or two molybdopterin (MPT) or molybdopterin dinucleotide cofactors

If the W sites of tungstoenzymes are structurally analogous to the Mo sites in molybdoenzymes, one might expect that molybdoenzymes would retain catalytic activity after substitution of Mo by W.

Mo-dependent organisms,when grown in the presence of tungstate, produce either inactive enzymes lacking any metal or W-substituted enzymes that have little or no catalytic activity

Hydrogenophaga pseudoflava (formerly Pseudomonas carboxydoflava is a carboxidotrophic bacterium capable of utilizing carbon monoxide (CO) as a source of carbon and energy under aerobic chemolithoautotrophic conditions

CO dehydrogenase : 2 mol Mo-molybdopterin cytosine dinucleotide (MCD) cofactor, 2 mol FAD, 8 mol iron and 8 mol acid-labile sulfur [240 kDa and an L2M2S2 subunit structure (L2 70 kDa, M2 33 kDa, S2 17 kDa)]

Tungstate inhibited chemolithoautotrophic growth of H. pseudoflava with CO, but had no effect on heterotrophic growth of the bacterium with pyruvate

Materials & Method1.Bacterial strain and growth conditions 1.Bacterial strain and growth conditions H.Pseudoflava (DSM 1084) was grown heterotrophically at 30℃ in a mH.Pseudoflava (DSM 1084) was grown heterotrophically at 30℃ in a m

ineral medium supplemented with 0.3% (w/v) pyruvate under gas miineral medium supplemented with 0.3% (w/v) pyruvate under gas mixture of 80% air and 20% CO at a flow rate of 31 minxture of 80% air and 20% CO at a flow rate of 31 min -1-1

2.Enzyme assays2.Enzyme assays The CO oxidation of CO dehydrogenase:1-pheny|-2-(4-The CO oxidation of CO dehydrogenase:1-pheny|-2-(4-

iodophenyl)-3-(4-nitrophenyl)-2H-tetrazoliuIn chbride(INT)/1-methoxyiodophenyl)-3-(4-nitrophenyl)-2H-tetrazoliuIn chbride(INT)/1-methoxyphenazime methosulfate (MPMS) as artincial electronphenazime methosulfate (MPMS) as artincial electronacceptors (using spectrophotometer)acceptors (using spectrophotometer)

Materials & Method 3.Enzyme purification3.Enzyme purification Oxic conditions at 4 C in 50 mM potassium phosphate,pH 7.2Oxic conditions at 4 C in 50 mM potassium phosphate,pH 7.2

(buffer A)(buffer A)FPLC (fast protein liquid chromatography) was employed for all chromatogrFPLC (fast protein liquid chromatography) was employed for all chromatographic pur|fication stepsiaphic pur|fication stepsiCell lysis : High pressure homogenizerCell lysis : High pressure homogenizer

Low spin centrifugation (crude extract)Low spin centrifugation (crude extract)Ultracentrifugation (cytoplasmic fraction)Ultracentrifugation (cytoplasmic fraction)Macroprep High Q Anion column (eluted bylinear gradient 0-1 MMacroprep High Q Anion column (eluted bylinear gradient 0-1 MKCl in buffer A)KCl in buffer A)

Ammonium su|fate(1.2 M)Ammonium su|fate(1.2 M)Butyl Sepharose 4 fast-flow hydrophobic interaction column(elutedButyl Sepharose 4 fast-flow hydrophobic interaction column(elutedby an decreasing linear gradient of 0.85-0 M ammonium sulfate inby an decreasing linear gradient of 0.85-0 M ammonium sulfate incombination with an increasing linear gradient of 0-30%combination with an increasing linear gradient of 0-30%isopropanol in buffer A)isopropanol in buffer A)Ultrafilteration (concentration)Ultrafilteration (concentration)Gel filteration on Sephadex G-25(desalting)Gel filteration on Sephadex G-25(desalting)

check fraction: CO dehydrogenase activitycheck fraction: CO dehydrogenase activity

5.Protein determination5.Protein determination Methods of BradfordMethods of Bradford

Homogeneous CO dehydrogenase was also quantined by its absorption at 450 nmHomogeneous CO dehydrogenase was also quantined by its absorption at 450 nm

6.Analysis of metals and acid-labile sulfur6.Analysis of metals and acid-labile sulfurIron : atomic absorption spectroscopy and colorimetrically by the formation of the Fe(IIron : atomic absorption spectroscopy and colorimetrically by the formation of the Fe(II)-ferrozine complexI)-ferrozine complexMo and W : inductively coupled plasma mass spectrometry(ICP-MS) and dithiol methodMo and W : inductively coupled plasma mass spectrometry(ICP-MS) and dithiol methodAcid-labile sulfur : methylene blue formationAcid-labile sulfur : methylene blue formation

7.Analysis of pterins,nucleotides and flavin7.Analysis of pterins,nucleotides and flavinPterm : Extraction of pterins from CO dehydrogenase with SDS and subsequent carboxPterm : Extraction of pterins from CO dehydrogenase with SDS and subsequent carboxamidomentylation with iodoacetamide.amidomentylation with iodoacetamide.HPLC, spectrophotometer,and spectrofluorometerHPLC, spectrophotometer,and spectrofluorometerFAD: extracted with SDS and ana|yzed by HPLC andFAD: extracted with SDS and ana|yzed by HPLC andspectrophotometerspectrophotometerNucleotides: released from MCD or FAD in CO dehydrogenase byNucleotides: released from MCD or FAD in CO dehydrogenase byhydrolysis with sulfuric acid for 1 0 min at 95℃,and then analyzedhydrolysis with sulfuric acid for 1 0 min at 95℃,and then analyzedby reverse-phase HPLCby reverse-phase HPLC

Materials & Method

8.Extraction and analysis of cytidine nucleotides8.Extraction and analysis of cytidine nucleotidesNucleotides were extracted from CO dehydrogenase by boiling for 90s in aqNucleotides were extracted from CO dehydrogenase by boiling for 90s in aqueous SDS, seperated from protein and SDS by ultrafiltration and analyzed ueous SDS, seperated from protein and SDS by ultrafiltration and analyzed by isocratic anion-exchange HPLCby isocratic anion-exchange HPLC

9.Analysis of MPT and MCD in crude extract9.Analysis of MPT and MCD in crude extractMPT and MCD in crude extracts were analyzed by conversion toMPT and MCD in crude extracts were analyzed by conversion toform A or form-A-CMPform A or form-A-CMPExtracts were adjusted to pH 2.5 and incubated overnight at 20℃ in the prExtracts were adjusted to pH 2.5 and incubated overnight at 20℃ in the presence of excess Iesence of excess I22 /KI.HPLC and spectrophotometer. /KI.HPLC and spectrophotometer.

10.Spectroscopic methods10.Spectroscopic methodsUltraviolet/visible spectrum-spectrophotometerUltraviolet/visible spectrum-spectrophotometerCD spectrum-spectropolarimeterCD spectrum-spectropolarimeterX-band EPR spectra.Brucker EMX spectrometer X-band EPR spectra.Brucker EMX spectrometer

Materials & Method

Result

Analysis of CO dehydrogenase species of H. pseudoflavagrown under different conditions of Mo and W supply by PAGE

CO dehydrogenase activity and contents

1g1g 에 에 protein protein 마다 녹아있는 금속의 양을 마다 녹아있는 금속의 양을 nmolnmol 로 환산로 환산 Mo Mo 이 이 WW 에 비하여 에 비하여 CO-DHCO-DH 상대적으로 상대적으로 affinity affinity 가 높다 가 높다

MPT MCD

Purification of CO dehydrogenase from H. pseudoflava

Iron-sulfur and Mo EPR spectra of purified active and inactive CO dehydrogenase species

2Fe:2S type II 2Fe:2S type I Mo

active

inactive

inactive

Identification of non-covalently bound cytidine nucleotides in Identification of non-covalently bound cytidine nucleotides in purified active and inactive CO dehydrogenase species.purified active and inactive CO dehydrogenase species.

inactive

active

CD spectra of purified active and inactive CO dehydrogenasespecie

active inactive Fe-S

Ultraviolet/visible spectra of purified active and inactive CO dehydrogenase species

DiscussionDiscussion

1.Repression of molybdate transport by tungstate.There was an inverse relationship between extracellular tungstate and intracellular Mo.

2.The effect of molybdate and tungstate on the biosynthesis, structure and redox centers of CO dehydrogenase.the biosyntheses of CoxL, CoxM, and CoxS,which resulted in an inactive enzyme, wereentirely independent of the metal

3.Role of Mo in the biosynthesis and integration of themolybdenum cofactor.Metal-free MPT, Mo-MPT or W-MPT complex would not be recognized by thecytidylyltransferase as an appropriate substrate for MCD synthesis

4.Anchoring of Mo-MCD to CO dehydrogenase.CO dehydrogenase displayed the highest affinity for CDP, indicating that the dinucleotidephosphates of MCD establish the strongest interactions of themolybdenum cofactor with the protein.