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

• Published in: PloS one 2020-08, Vol.15 (8), p.e0231560-e0231560
• Main Authors: Shmonova, Ekaterina A, Voloshina, Olga V, Ovsienko, Maksim V, Smirnov, Sergey V, Nolde, Dmitry E, Doroshenko, Vera G
• Format: Article
• Language: English
• Subjects: 4-Hydroxyphenylpyruvate dioxygenase; Actinomycetes; Biology and Life Sciences; Biosynthesis; Biotechnology; Carbon dioxide; Chemical properties; Chromosomes; Cloning; Cobalt; Cofactors; Corynebacterium glutamicum; Corynebacterium glutamicum - enzymology; Corynebacterium glutamicum - genetics; Dehydration; Deoxyribonucleic acid; Dimerization; Dimers; DNA; DNA polymerase; DNA, Recombinant - genetics; E coli; Enzymes; Escherichia coli - metabolism; Fermentation; Genetic aspects; Glucose; Glycerol; Hydro-Lyases - chemistry; Hydro-Lyases - genetics; Hydro-Lyases - metabolism; Hydrogen-Ion Concentration; Hydroxybenzoates - metabolism; Hydroxybenzoic acids; Identification and classification; Magnesium; Medicine and Health Sciences; Microbial enzymes; Microorganisms; Models, Molecular; Oligomerization; Physical Sciences; Physiological aspects; Plasmids; Production processes; Protein Domains; Protein Multimerization; Protein Structure, Quaternary; Proteins; Protocatechuic acid; Pseudomonas putida; Structural stability; Structure
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0231560

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): Co2+, Mg2+, Mn2+. The Km and kcat values for the QsuB enzyme (Km ~ 1 mM, kcat ~ 61 s-1) were two and three times higher than those for N-QsuB. 3,4-DHBA inhibited QsuB (Ki ~ 0.38 mM, Ki' ~ 0.96 mM) and N-QsuB (Ki ~ 0.69 mM) enzymes via mixed and noncompetitive inhibition mechanism, respectively. E. coli MG1655ΔaroEPlac‒qsuB strain produced three times more 3,4-DHBA from glucose in test tube fermentation than the MG1655ΔaroEPlac‒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).