Isolation and properties of naphthoate synthetase from Mycobacterium phlei

Cell-free extracts obtained by sonication of Mycobacterium phlei cells contain an important enzyme of the menaquinone (= vitamin K₂) biosynthetic pathway. This enzyme, naphthoate synthetase (1,4-dihydroxy-2-naphthoate synthetase), was partially purified by chromatography on Sepharose 6BCL. Conversion of o-succinylbenzoate to 1,4-dihydroxy-2-naphthoate was followed by a radioactivity assay using o-[2,3-¹⁴C₂]succinylbenzoate, or by a spectrophotofluorometric assay. o-[1-¹³C]Succinylbenzoate was converted intact by the extracts to dihydroxynaphthoate containing ¹³C only in the carboxyl carbon atom. For maximum activity, the enzyme requires ATP, Mg²⁺, and coenzyme A. The pH optimum is 6.9 and the molecular weight approximately 44,000. In the presence of farnesyl pyrophosphate, the extracts convert o-[2,3-¹⁴C₂]succinylbenzoate to ¹⁴C-containing menaquinone.