Biosynthesis of o-succinylbenzoic acid. II: Properties of o-succinylbenzoic acid synthase, an enzyme involved in vitamin K2 biosynthesis.
The enzyme system (OSB2 synthase) catalyzing the synthesis of o-succinylbenzoic acid from isochorismic acid and alpha-ketoglutaric acid in the presence of thiamine pyrophosphate was isolated from Escherichia coli AN 154 and characterized. The purification factor of the enzyme did not increase during column chromatography on Sephadex G-200 or chromatofocusing, suggesting that the OSB synthase is labile. Chromatography on DEAE-Sephadex A-50 or A-25 showed that an enzyme activity separated from fractions containing OSB synthase that decarboxylates alpha-ketoglutarate. This activity is provisionally referred to as the decarboxylating "subunit" or decarboxylating activity of OSB synthase. Both the "subunit" and the holoenzyme were characterized with respect to pH optimum, temperature optimum, and KM values. The OSB synthase loses all activity during treatment with EDTA and activity is most efficiently restored with Mn2+. The activity of the decarboxylating subunit did not depend on Mn2+. When the decarboxylating fraction was incubated with alpha-ketoglutarate and thiamine pyrophosphate, succinic semialdehyde could be isolated as its hydrazone. After treatment of the incubation mixture with phosphorylase a compound was isolated which is most likely the thiamine adduct of succinic semialdehyde.
Weische
A
A
Garvert
W
W
Leistner
E
E
D019755:Carbon-Carbon Lyases; D002384:Catalysis; D002845:Chromatography / Q000379:methods; D003653:Decarboxylation; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D008970:Molecular Weight; D010654:Phenylbutyrates / Q000096:biosynthesis*; D013696:Temperature; D013831:Thiamine / Q000378:metabolism; D014812:Vitamin K / Q000096:biosynthesis
D007656:Ketoglutaric Acids; D010654:Phenylbutyrates; D014812:Vitamin K; C013691:o-succinylbenzoic acid; D008190:Lyases; D019755:Carbon-Carbon Lyases; C052986:o-succinylbenzoic acid synthase; D013831:Thiamine