The overexpression and complete amino acid sequence of Escherichia coli 3-dehydroquinase

Abstract

The enzyme 3-dehydroquinase was purified in milligram quantities from an overproducing strain of Escherichia coli. The amino acid sequence was deduced from the nucleotide sequence of the aroD gene and confirmed by determining the amino acid composition of the overproduced enzyme and its N-terminal amino acid sequence. The complete polypeptide chain consists of 240 amino acid residues and has a calculated subunit Mr of 26,377. Transcript mapping revealed that aroD is a typical monocistronic gene.

Identification of the active-site lysine residues of two biosynthetic 3-dehydroquinases

“…Only CNBr digestion of this enzyme was performed. As the complete amino acid sequence of E. coli 3-dehydroquinase is available from the DNA sequence of the aroD gene, which encodes this enzyme (Duncan et al, 1986; also the present paper), we were not only able to predict the exact number of CNBr-cleavage fragments that would be produced, but also their precise size and composition. We expected to find 11 peptides, ranging in size from two to 47 amino acid residues.…”

“…Subsequent digestion of the labelled protein and isolation and characterization of labelled peptides has allowed us to determine the sequence around the active-site lysine residue in 3-dehydroquinase from two species, N. crassa and E. coli. A complete nucleotide sequence for the E. coli aroD gene has been published (Duncan et al, 1986). Subsequent DNAsequencing work in our laboratory has revealed that this sequence contained errors at four locations.…”

“…Clearly all four sequences are related. One notable feature of the revised E. coli 3-dehydroquinase sequence is the absence of cysteine residues; the original sequence had predicted that the protein would contain three cysteine residues E. coli S. cerevisiae A. nidulans E. coli S. cerevisiae A. nidulans E. coli S. cerevisiae A. nidulans N. crassa (Duncan et al, 1986). This means that the reduction and alkylation step used in the work-up of the active-site-modified E. coli enzyme was not necessary.…”

“…This means that the reduction and alkylation step used in the work-up of the active-site-modified E. coli enzyme was not necessary. The availability of an overproducing strain of E. coli has made possible the isolation of 100 mg quantities of 3-dehydroquinase from this organism (Duncan et al, 1986). We have recently improved enzyme yields considerably by constructing a secondgeneration overproducer, which relies not on copy number but on placement of aroD behind the inducible and very powerful tac promoter (K. Duncan, unpublished work).…”

“…Members of three classes catalyse the biosynthetic reaction described above, and illustrate the diversity found in the organization of the shikimate pathway enzymes and genes in various organisms. Monofunctional 3-dehydroquinases have been identified in bacteria (Berlyn & Giles, 1969); the Escherichia coli enzyme has been purified to homogeneity and the complete sequence is known Duncan et al, 1986). The plant enzyme occurs on a bifunctional polypeptide chain in association with shikimate dehydrogenase (Polley, 1978;Koshiba, 1978;Mousdale et al, 1987), which catalyses the fourth step on the shikimate pathway.…”