Molecular microbiology Lab. 한승정

1. 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 electronacceptors (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 stepsiCell lysis : High pressure homogenizer Low spin centrifugation (crude extract)Ultracentrifugation (cytoplasmic fraction)Macroprep High Q Anion column (eluted bylinear gradient 0-1 MKCl in buffer A) Ammonium su|fate(1.2 M)Butyl Sepharose 4 fast-flow hydrophobic interaction column(elutedby an decreasing linear gradient of 0.85-0 M ammonium sulfate incombination 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 BradfordHomogeneous CO dehydrogenase was also quantined by its absorption at 450 nm 6.Analysis of metals and acid-labile sulfurIron : atomic absorption spectroscopy and colorimetrically by the formation of the Fe(II)-ferrozine complexMo and W : inductively coupled plasma mass spectrometry(ICP-MS) and dithiol methodAcid-labile sulfur : methylene blue formation 7.Analysis of pterins,nucleotides and flavinPterm : Extraction of pterins from CO dehydrogenase with SDS and subsequent carboxamidomentylation with iodoacetamide.HPLC, spectrophotometer,and spectrofluorometerFAD: extracted with SDS and ana|yzed by HPLC andspectrophotometerNucleotides: released from MCD or FAD in CO dehydrogenase byhydrolysis with sulfuric acid for 1 0 min at 95℃,and then analyzedby reverse-phase HPLC

8. Materials & Method 8.Extraction and analysis of cytidine nucleotidesNucleotides 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 extractMPT and MCD in crude extracts were analyzed by conversion toform A or form-A-CMPExtracts were adjusted to pH 2.5 and incubated overnight at 20℃ in the presence of excess I2 /KI.HPLC and spectrophotometer. 10.Spectroscopic methodsUltraviolet/visible spectrum-spectrophotometerCD spectrum-spectropolarimeterX-band EPR spectra.Brucker EMX spectrometer

9. Result

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

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

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 active inactive

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.