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. Abstract.
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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.
1) Subunit structure
3) Mo-MCD cofactor : biosynthesis and its insertion
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
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)
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
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.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
Result dehydrogenase and the molybdopterin cytosine dinucleotide-type of molybdenum cofactor in
2Fe:2S type II
2Fe:2S type I
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
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.