Purification and characterization of a dual function 3-dehydroquinate dehydratase from Amycolatopsis methanolica

• Published in: Journal of general microbiology Vol. 138; no. 11; pp. 2449 - 2457
• Main Authors: Euverink, G.J.W, Hessels, G.I, Vrijbloed, J.W, Coggins, J.R, Dijkhuizen, L
• Format: Journal Article
• Language: English
• Published: England Soc General Microbiol 01.11.1992
• Subjects: 3-dehydroquinate dehydratase; Actinomycetales; Actinomycetales - enzymology; actinomycetes; Amino Acid Sequence; amino acid sequences; Amycolatopsis methanolica; aromatic amino acids; biochemical pathways; characterization; enzyme activity; hydro-lyases; Hydro-Lyases - isolation & purification; Kinetics; metabolism; Molecular Sequence Data; Molecular Weight; Mutagenesis; mutants; physicochemical properties; protein synthesis; purification; quinic acid; Quinic Acid - analogs & derivatives; Quinic Acid - metabolism; sequence alignment; Sequence Homology, Amino Acid; shikimic acid; Shikimic Acid - metabolism; structure-activity relationships; transcription (genetics)
• ISSN: 0022-1287
• DOI: 10.1099/00221287-138-11-2449

Studies on hydroaromatic metabolism in the actinomycete Amycolatopsis methanolica revealed that the organism grows rapidly on quinate (but not on shikimate) as sole carbon- and energy source. Quinate is initially converted into the shikimate pathway intermediate 3-dehydroquinate by an inducible NAD+-dependent quinate/shikimate dehydrogenase. 3-Dehydroquinate dehydratase subsequently converts 3-dehydroquinate into 3-dehydroshikimate, which is used partly for the biosynthesis of aromatic amino acids, and is partly catabolized via protocatechuate and the beta-ketoadipate pathway. Enzyme studies and analysis of mutants clearly showed that the single 3-dehydroquinate dehydratase present in A. methanolica has a dual function, the first example of a 3-dehydroquinate dehydratase enzyme involved in both the catabolism of quinate and the biosynthesis of aromatic amino acids. This enzyme was purified over 1700-fold to homogeneity. Its further characterization indicated that it is a Type II 3-dehydroquinate dehydratase, a thermostable enzyme with a large oligomeric structure (native Mr 135 X 10(3)) and a subunit Mr of 12 X 10(3). Characterization of aromatic amino acid auxotrophic mutants of A. methanolica suggested that genes encoding 3-dehydroquinate synthase and 3-dehydroquinate dehydratase are genetically linked but their transcription results in the synthesis of two separate proteins.