Purification and characterization of an NAD(+)-linked formaldehyde dehydrogenase from the facultative RuMP cycle methylotroph Arthrobacter P1.
When Arthrobacter P1 is grown on choline, betaine, dimethylglycine or sarcosine, an NAD(+)-dependent formaldehyde dehydrogenase is induced. This formaldehyde dehydrogenase has been purified using ammonium sulphate fractionation, anion exchange- and hydrophobic interaction chromatography. The molecular mass of the native enzyme was 115 kDa +/- 10 kDa. Gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the molecular mass of the subunit was 56 kDa +/- 3 kDa, which is consistent with a dimeric enzyme structure. After ammonium sulphate fractionation the partially purified enzyme required the addition of a reducing reagent in the assay mixture for maximum activity. The enzyme was highly specific for its substrates and the Km values were 0.10 and 0.80 mM for formaldehyde and NAD+, respectively. The enzyme was heat-stable at 50 degrees C for at least 10 min and showed a broad pH optimum of 8.1 to 8.5. The addition of some metal-binding compounds and thiol reagents inhibited the enzyme activity.
Attwood
M M
MM
Department of Microbiology, University of Sheffield, UK.
Arfman
N
N
Weusthuis
R A
RA
Dijkhuizen
L
L
D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000447:Aldehydes / Q000378:metabolism; D001173:Arthrobacter / Q000201:enzymology*; D002614:Chelating Agents / Q000494:pharmacology; D004795:Enzyme Stability; D005557:Formaldehyde / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000447:Aldehydes; D002614:Chelating Agents; D013439:Sulfhydryl Reagents; D009243:NAD; D005557:Formaldehyde; D000445:Aldehyde Oxidoreductases; C087006:formaldehyde dehydrogenase, glutathione-independent