1. Molecular microbiology Lab. 한승정
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.
한승정

2. Abstract
The molybdenum-containing iron-sulfur flavoprotein CO dehydrogenase is expressed 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: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.

3. Introduction 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

4. 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

5. Materials & Method 1.Bacterial strain and growth conditions
H.Pseudoflava (DSM 1084) was grown heterotrophically at 30℃ in a mineral medium supplemented with 0.3% (w/v) pyruvate under gas mixture of 80% air and 20% CO at a flow rate of 31 min-1
2.Enzyme assays
The CO oxidation of CO dehydrogenase:1-pheny|-2-(4- iodophenyl)-3-(4-nitrophenyl)-2H-tetrazoliuIn chbride(INT)/1-methoxyphenazime methosulfate (MPMS) as artincial electron acceptors (using spectrophotometer)

6. Materials & Method 3.Enzyme purification
Oxic conditions at 4 C in 50 mM potassium phosphate,pH 7.2 (buffer A) FPLC (fast protein liquid chromatography) was employed for all chromatographic pur|fication stepsi Cell lysis : High pressure homogenizer
Low spin centrifugation (crude extract) Ultracentrifugation (cytoplasmic fraction) Macroprep High Q Anion column (eluted bylinear gradient 0-1 M KCl in buffer A)
Ammonium su|fate(1.2 M) Butyl Sepharose 4 fast-flow hydrophobic interaction column(eluted by an decreasing linear gradient of M ammonium sulfate in combination with an increasing linear gradient of 0-30% isopropanol in buffer A) Ultrafilteration (concentration) Gel filteration on Sephadex G-25(desalting) check fraction: CO dehydrogenase activity

7. Materials & Method 5.Protein determination
Methods of Bradford Homogeneous CO dehydrogenase was also quantined by its absorption at 450 nm
6.Analysis of metals and acid-labile sulfur Iron : atomic absorption spectroscopy and colorimetrically by the formation of the Fe(II)-ferrozine complex Mo and W : inductively coupled plasma mass spectrometry(ICP-MS) and dithiol method Acid-labile sulfur : methylene blue formation
7.Analysis of pterins,nucleotides and flavin Pterm : Extraction of pterins from CO dehydrogenase with SDS and subsequent carboxamidomentylation with iodoacetamide. HPLC, spectrophotometer,and spectrofluorometer FAD: extracted with SDS and ana|yzed by HPLC and spectrophotometer Nucleotides: released from MCD or FAD in CO dehydrogenase by hydrolysis with sulfuric acid for 1 0 min at 95℃,and then analyzed by reverse-phase HPLC

8. Materials & Method
8.Extraction and analysis of cytidine nucleotides Nucleotides were extracted from CO dehydrogenase by boiling for 90s in aqueous SDS, seperated from protein and SDS by ultrafiltration and analyzed by isocratic anion-exchange HPLC
9.Analysis of MPT and MCD in crude extract MPT and MCD in crude extracts were analyzed by conversion to form A or form-A-CMP Extracts were adjusted to pH 2.5 and incubated overnight at 20℃ in the presence of excess I2 /KI.HPLC and spectrophotometer.
10.Spectroscopic methods Ultraviolet/visible spectrum-spectrophotometer CD spectrum-spectropolarimeter X-band EPR spectra.Brucker EMX spectrometer

9. Result

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

11. CO dehydrogenase activity and contents
MPT
MCD
1g에 protein 마다 녹아있는 금속의 양을 nmol로 환산
Mo 이 W에 비하여 CO-DH상대적으로 affinity 가 높다

12. Purification of CO dehydrogenase from H. pseudoflava

13. 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

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

15. CD spectra of purified active and inactive CO dehydrogenasespecie
Fe-S

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

17. Discussion 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, were
entirely independent of the metal
3.Role of Mo in the biosynthesis and integration of the
molybdenum cofactor.
Metal-free MPT, Mo-MPT or W-MPT complex would not be recognized by the
cytidylyltransferase 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 the
molybdenum cofactor with the protein.