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

A novel plasmid DNA construct has been developed for use in the synthe sis of a histidine biosynthesis ospho-beta-D-ribosyl)formimino]-5-amin oimidazole-4 carboxamide ribonucleotide (5'-ProFAR, 1). The approach a llows for a convenient, millimole-scale preparation of the bis-ribosyl nucleotide and provides material of suitable purity for spectral anal yses and biochemical studies. A highly enriched source of the first th ree enzymes in the histidine biosynthetic pathway has been developed b y constructing a recombinant DNA plasmid which directs the overexpress ion of these proteins. When used in a strain of Escherichia ia coil th at is devoid of other histidine biosynthetic enzymes, cell-free extrac ts can be coupled with 5-phospho-D-ribosyl alpha-1-pyrophosphate synth etase 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.