The overall aim of these investigations is to understand in detail the means by which microorganisms synthesize vitamin K. This fat soluble vitamin plays a significant role in human health; vitamin K responsive hypoprothrombinemia is an important clinical problem under a number of conditions. The biosynthesis of this vitamin by microorganisms is of especial interest since, under normal physiological conditions, the bacterial flora of the human interest may contribute from 50-100% of the vitamin K requirement. The main emphasis of the work is on the biosynthetic pathway which assembles the aromatic, naphthoquinone nucleus of this vitamin. It has been known for some time that this nucleus drives from reactions of the """"""""shikimate pathway"""""""", and more recently, isochorismate has been identified as the starting point for assembly of the naphthoquinone. Intermediates which have now been identified are 2-succinyl-6-hydroxy-2, 4-cyclohexadiene-1- carboxylate (SHCHC), o-succinylbenzoate (OSB), a CoA ester of OSB (OSB-CoA), 1, 4-dihydroxy-2-naphthoate (DHNA), and desmethylmenaquinone (DMK). The various enzymes of this biosynthetic pathway will be isolated, purified, and examined to determine how they are regulated. In several cases, since only low levels of enzymes are available, the construction of bacterial strains with gene amplification will be undertaken; overexpression of the gene should provide adequate amounts of protein for detailed study. Much of the work will focus on menaquinone (vitamin K2) in Escherichia coli; however, the biosynthetic pathway will also be investigated in Euglena gracilis.
