Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and properties of o-succinylbenzoyl-coenzyme A synthetase from Escherichia coli.
The coenzyme A (CoA)- and ATP-dependent conversion of o-succinylbenzoic acid [OSB; 4-(2'-carboxyphenyl)-4-oxobutyric acid], to o-succinylbenzoyl-CoA is carried out by the enzyme o-succinylbenzoyl-CoA synthetase. o-Succinylbenzoyl-CoA is a key intermediate in the biosynthesis of menaquinone (vitamin K2) in both gram-negative and gram-positive bacteria. The enzyme has been overexpressed and purified to homogeneity. The purified enzyme was found to have a native molecular mass of 185 kDa as determined by gel filtration column chromatography on Sephacryl S-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis established a subunit molecular mass of 49 kDa. Thus, the enzyme is a homotetramer. The enzyme showed a pH optimum of 7.5 to 8.0 and a temperature optimum of 30 to 40 degrees C. The Km values for OSB, ATP, and CoA were 16, 73.5, and 360 microM, respectively. Of the various metal ions tested, Mg2+ was found to be the most effective in stimulating the enzyme activity. Studies with substrate analogs showed that neither benzoic acid nor benzoylpropionic acid (succinylbenzene) is a substrate for the enzyme. Thus, it appears that both the benzoyl carboxyl group and the succinyl side chain are required for activation of the aliphatic carboxyl group.
Kwon
O
O
Department of Biological Sciences, Northern Illinois University, DeKalb 60115-2861, USA.
Bhattacharyya
D K
DK
Meganathan
R
R
D001565:Benzoates / Q000378:metabolism; D002412:Cations / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013389:Succinate-CoA Ligases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D014169:Transformation, Bacterial; D014812:Vitamin K / Q000096:biosynthesis*
D001565:Benzoates; D002412:Cations; D014812:Vitamin K; D013389:Succinate-CoA Ligases; C022693:O-succinylbenzoate - CoA ligase; D008274:Magnesium