A gene encoding dehydroquinate dehydratase (DHQase) was cloned from Streptomyces hygroscopicus var. ascomyceticus. The 528-bp open reading frame specified a primary translation product of 175 amino acids with a calculated Mr of 18,789. The predicted amino acid sequence of the DHQase showed similarities to bacterial and fungal type II DHQases. Overexpression of the dhq gene was accomplished in Escherichia coli using a gene fusion technique in which a malE, the gene encoding the maltose binding protein (MBP), was fused via a short oligonucleotide region to the beginning of dhq. The recombinant MBP-DHQase fusion protein was purified by affinity chromatography and cleaved using thrombin. The resulting DHQase, separated from the MBP, demonstrated typical properties of a type II DHQase: a relatively high Km for the dehydroquinate substrate (650 microM) and extreme thermal stability. The subunit Mr estimated by SDS-PAGE was 19,000, and the native Mr estimated by gel-exclusion chromatography and sucrose-density centrifugation was 130,000, suggesting that the enzyme is a homoheptamer (type II DHQases are typically homododecamers). The MBP-DHQase complex also adopted a heptameric structure and was a thermostable, fully active DHQase, indicating that the N-terminus is not involved in formation of protomer-protomer complexes. Previous analyses have supported positioning the N-terminus of type II DHQases close to the active site and a conformational change in this region coincident with ligand binding. Nonetheless, the Km and relative kcat obtained for MBP-DHQase were indistinguishable from those observed for DHQase. Inactivation data of the DHQase from S. hygroscopicus with the arginine-specific reagent phenylglyoxal showed that a modified Arg residue(s) is likely close to the N-terminus and active site of DHQase, but does not play an essential role in catalysis.
Copyright 1998 Academic Press.