A PLASMID-BASED APPROACH FOR THE SYNTHESIS OF A HISTIDINE BIOSYNTHETIC INTERMEDIATE

A novel plasmid DNA construct has been developed for use in the synthesis of a histidine biosynthesis intermediate,N-1-C(5''-phospho-beta-D-ribosyl)formimino]-5-aminoimidazole-4 carboxamide ribonucleotide (5'-ProFAR, 1). The approach allows for a convenient, millimole-scale preparation of the bis-ribosyl nucleotide and provides material of suitable purity for spectral analyses and biochemical studies. A highly enriched source of the first three enzymes in the histidine biosynthetic pathway has been developed by constructing a recombinant DNA plasmid which directs the overexpression of these proteins. When used in a strain of Escherichia ia coil that is devoid of other histidine biosynthetic enzymes, cell-free extracts can be coupled with 5-phospho-D-ribosyl alpha-1-pyrophosphate synthetase and an ATP recycling system to prepare 1 directly from D-ribose 5-phosphate. This paper also presents the first spectral analysis of 5'-ProFAR and its major decomposition products.