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~6 1 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 MG1655ΔaroEP lac-qsuB strain produced three times more 3,4-DHBA from glucose in test tube fermentation than the MG1655ΔaroEP 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).

The production of 3,4-dihydroxybenzoic acid (3,4-DHBA or protocatechuate) is a relevant task owing to 3,4-DHBA’s pharmaceutical properties and its use as a precursor for subsequent synthesis of high value-added chemicals. The microbial production of 3,4-DHBA using dehydroshikimate dehydratase (DSD) (EC: 4.2.1.118) has been demonstrated previously. DSDs from soil-dwelling organisms (where DSD is involved in quinate/shikimate degradation) and from Bacillus spp. (synthesizing the 3,4-DHBA-containing siderophore) were compared in terms of the kinetic properties and their ability to produce 3,4-DHBA. Catabolic DSDs from Corynebacterium glutamicum (QsuB) and Neurospora crassa (Qa-4) had higher Km (1 and 0.6 mM, respectively) and kcat (61 and 220 s−1, respectively) than biosynthetic AsbF from Bacillus thuringiensis (Km~0.04 mM, kcat~1 s−1). Product inhibition was found to be a crucial factor when choosing DSD for strain development. AsbF was more inhibited by 3,4-DHBA (IC50~0.08 mM), and Escherichia coli MG1655 ΔaroE PlacUV5-asbFattφ80 strain provided only 0.2 g/L 3,4-DHBA in test-tube fermentation. Isogenic strains MG1655 ΔaroE PlacUV5-qsuBattφ80 and MG1655 ΔaroE PlacUV5-qa-4attφ80 expressing QsuB and Qa-4 with IC50 ~0.35 mM and ~0.64 mM, respectively, accumulated 2.7 g/L 3,4-DHBA under the same conditions.

Corynebacterium glutamicum AJ1511 and Escherichia coli BW25113 strains were compared in terms of resistance to sarcosine (N-methylglycine). The E. coli strain was more sensitive to sarcosine than C. glutamicum especially when grown in minimal medium. Growth inhibition of the BW25113 strain in minimal M9 medium containing 0.5 M sarcosine was overcome by the addition of glycine. Inactivation of the glycine cleavage (GCV) system (∆gcvP) as well as the removal of its activator (∆gcvA) in BW25113 cells increased the threshold for sarcosine inhibition up to 0.75 M. Activation of the promoter of the E. coli gcvTHP operon by 0.1–0.4 M sarcosine added to M9 medium was demonstrated in vivo using dasherGFP as the reporter. Sensitivity to sarcosine on minimal glucose medium is suggested to be a character of gram-negative bacteria with GcvA/GcvR regulation of the GCV system.

2 The dehydroshikimate dehydratase (DSD) from Corynebacterium glutamicum 3 encoded by the qsuB gene is related to the previously described QuiC1 protein 4 (39.9% identity) from Pseudomonas putida. QuiC1 and QsuB are both two-domain 5 bacterial DSDs. The N-terminal domain provides dehydratase activity, while the 6 C-terminal domain has sequence identity with 4-hydroxyphenylpyruvate 7 dioxygenase. Here, the QsuB protein and its DSD domain (N-QsuB) were 8 expressed in the T7 system, purified and characterized. QsuB was present mainly 9 in octameric form (60%), while N-QsuB had a predominantly monomeric structure 10 (80%) in solution. Both proteins possessed DSD activity with one of the following 11 cofactors (listed in order of decreasing activity): Co 2+ , Mg 2+ , Mn 2+ or Ca 2+ . The K m 12 and k cat values for QsuB were two and three times higher, respectively (K m ~ 1 13 mM, k cat ~ 61 s -1 ) than those for N-QsuB. Notably, 3,4-DHBA inhibited both 14 enzymes via an uncompetitive mechanism. QsuB and N-QsuB were tested for 3,4-15 DHBA production from glucose in E. coli. MG1655aroE P lac qsuB produced at 16 least two times more 3,4-DHBA than MG1655aroE P lac n-qsuB in the presence 17 of isopropyl β-D-1-thiogalactopyranoside.18