Open AccessCharacterization of the Corynebacterium glutamicum dehydroshikimate dehydratase QsuB and its potential for microbial production of protocatechuic acid
Author(s) -
Ekaterina A. Shmonova,
O. V. Voloshina,
М. В. Овсиенко,
Sergey V. Smirnov,
Dmitry E. Nolde,
V. G. Doroshenko
Publication year - 2020
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0231560
Subject(s) - dehydratase , pseudomonas putida , corynebacterium glutamicum , enzyme kinetics , enzyme , biochemistry , fermentation , biology , enzyme assay , chemistry , stereochemistry , active site , gene
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 . Both QuiC1 and QsuB are 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 N-terminal 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 aqueous buffer. Both proteins possessed DSD activity with one of the following cofactors (listed in the order of decreasing activity): Co 2+ , Mg 2+ , Mn 2+ . The K m and k cat values for the QsuB enzyme (K m ~ 1 mM, k cat ~ 61 s -1 ) were two and three times higher than those for N-QsuB. 3,4-DHBA inhibited QsuB (K i ~ 0.38 mM, K i ’ ~ 0.96 mM) and N-QsuB (K i ~ 0.69 mM) enzymes via mixed and noncompetitive inhibition mechanism, respectively. E . coli MG1655Δ aroE P lac ‒ qsuB strain produced three times more 3,4-DHBA from glucose in test tube fermentation than the MG1655Δ aroE P lac ‒ n - qsuB strain. The C-terminal domain activity towards 3,4-DHBA was not established in vitro . This domain was proposed to promote protein oligomerization for maintaining structural stability of the enzyme. The dimer formation of QsuB protein was more predictable (ΔG = ‒15.8 kcal/mol) than the dimerization of its truncated version N-QsuB (ΔG = ‒0.4 kcal/mol).