Characterization of the Corynebacterium glutamicum dehydroshikimate dehydratase QsuB and its potential for microbial production of protocatechuic acid

Abstract

The dehydroshikimate dehydratase (DSD) from Corynebacterium glutamicum encoded by the qsuB gene is related to the previously described QuiC1 protein (39.9% identity) from Pseudomonas putida. QuiC1 and QsuB are both two-domain bacterial DSDs. The N-terminal domain provides dehydratase activity, while the C-terminal domain has sequence identity with 4-hydroxyphenylpyruvate dioxygenase. Here, the QsuB protein and its DSD domain (N-QsuB) were expressed in the T7 system, purified and characterized. QsuB was present mainly in octameric form (60%), while N-QsuB had a predominantly monomeric structure (80%) in solution. Both proteins possessed DSD activity with one of the following cofactors (listed in order of decreasing activity): Co2+, Mg2+, Mn2+ or Ca2+. The Km and kcat values for QsuB were two and three times higher, respectively (Km ~ 1 mM, kcat ~ 61 s-1) than those for N-QsuB. Notably, 3,4-DHBA inhibited both enzymes via an uncompetitive mechanism. QsuB and N-QsuB were tested for 3,4-DHBA production from glucose in E. coli. MG1655{Delta}aroE Plac-qsuB produced at least two times more 3,4-DHBA than MG1655{Delta}aroE Plac-n-qsuB in the presence of isopropyl {beta}-D-1-thiogalactopyranoside.