EXPRESSION OF RAT-LIVER TRYPTOPHAN 2,3-DIOXYGENASE IN ESCHERICHIA-COLI - STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF THE PURIFIED ENZYME

The hepatic hemoprotein tryptophan 2,3-dioxygenase (TDO) is the key re gulatory enzyme that, through irreversible degradation, controls the f lux of tryptophan through physiologically relevant pathways. This enzy me is composed of four identical subunits and in its fully assembled t etrameric form requires 2 mol of heme (Fe+2-protoporphyrin IX)/mol of protein for functional competence. Using a full-length cDNA for the ra t liver TDO subunit (pUC119/TDO) as the template, TDO cDNA was amplifi ed by polymerase chain reaction (PCR) and incorporated into the expres sion vector pTrc99A after introduction of convenient restriction sites as well as modification of the second codon AGT to GCT to optimize it s bacterial expression. DH5 alpha F' strain Escherichia coli cells tra nsfected with this pTrc99A/TDO construct expressed soluble, functional ly active, tetrameric TDO protein in high yields. The enzyme was isola ted from 30,000g supernatant of cellysates, purified by ion-exchange c hromatography, and its spectral and catalytic properties were assessed in terms of its substrate and prosthetic moiety specificities, in alm ost all aspects, the bacterially expressed enzyme was found to be iden tical to that of the rat liver. Heterologous expression of the fully f unctional enzyme, we trust, will enable future elucidation of its stru cture-function relationships. (C) 1996 Academic Press, Inc.