AbstractThe 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. MG1655ΔaroE Plac–qsuB produced at least two times more 3,4-DHBA than MG1655ΔaroE Plac–n-qsuB in the presence of isopropyl β-D-1-thiogalactopyranoside.
Characterization of the Corynebacterium glutamicum dehydroshikimate dehydratase QsuB and its potential for microbial production of protocatechuic acid