Menaquinone Biosynthesis: Mechanistic Studies on 5,8-Dihydroxy-2-Naphthoate Synthase and Dehypoxanthine Futalosine Cyclase

Abstract

This work details mechanistic studies of two enzymes in the futalosine dependent biosynthetic pathway of menaquinone (vitamin K), 5,8-Dihydroxy-2- naphthoate synthase (MqnD) and dehypoxanthine futalosine cyclase (MqnC). MqnD catalyzes the conversion of cyclic dehypoxanthine futalosine to 5,8-dihydroxy-2- naphthoic acid and an uncharacterized product. The main limitation in the study of this enzyme was the lack of substrate availability due to either difficult chemical synthesis or poor activity of an upstream enzyme in this pathway.

This study describes a chemoenzymatic synthesis of cyclic dehypoxanthine futalosine. This synthesis achieved a 2-fold yield enhancement by using titanium(III) citrate as the reducing agent and another 5-fold yield enhancement using a fluorinated analog of dehypoxanthine futalosine which was converted to cyclic dehypoxanthine futalosine by an ipso substitution mechanism. This synthetic route enabled the synthesis of cyclic dehypoxanthine futalosine in sufficient quantity to identify the second reaction product and to determine that the MqnD-catalyzed reaction proceeds by a hemiacetal ring opening-tautomerization-retroaldol sequence.

The radical S-Adenosylmethionine (SAM) enzyme, MqnC, found in the alternative menaquinone biosynthetic pathway catalyzes the conversion of dehypoxanthine futalosine to cyclic dehypoxanthine futalosine. This work reports the second successful use of an SRN1 fragmentation to trap an aryl radical anion intermediate, the use of a reducing agent gradient to pinpoint the branch point of the SRN1 fragmentation, and further work in understanding an unexpected 5-fold product enhancement when using a fluorinated substrate analog.