Characterization of shikimate dehydrogenase homologues of Corynebacterium glutamicum

Abstract

The function of three Corynebacterium glutamicum shikimate dehydrogenase homologues, designated as qsuD (cgR_0495), cgR_1216, and aroE (cgR_1677), was investigated. A disruptant of aroE required shikimate for growth, whereas a qsuD-deficient strain did not grow in medium supplemented with either quinate or shikimate as sole carbon sources. There was no discernible difference in growth rate between wild-type and a cgR_1216-deficient strain. Enzymatic assays showed that AroE both reduced 3-dehydroshikimate, using NADPH as cofactor, and oxidized shikimate, the reverse reaction, using NADP(+) as cofactor. The reduction reaction was ten times faster than the oxidation. QsuD reduced 3-dehydroquinate using NADH and oxidized quinate using NAD(+) as cofactor. Different from the other two homologues, the product of cgR_1216 displayed considerably lower enzyme activity for both the reduction and the oxidation. The catalytic reaction of QsuD and AroE was highly susceptible to pH. Furthermore, reduction of 3-dehydroshikimate by AroE was inhibited by high concentrations of shikimate, but neither quinate nor aromatic amino acids had any effect on the reaction. Expression of qsuD mRNA was strongly enhanced in the presence of shikimate, whereas that of cgR_1216 and aroE decreased. We conclude that while AroE is the main catalyst for shikimate production in the shikimate pathway, QsuD is essential for quinate/shikimate utilization.

Structurally diverse dehydroshikimate dehydratase variants participate in microbial quinate catabolism

“…Due to shared usage of the substrate, dehydroshikimate, DSDs may compete with the chorismate biosynthesis-associated SDH, AroE (Tresguerres et al, 1972). The AroE enzyme from C. glutamicum (an organism which also encodes an ortholog of QuiC1) exhibits a k cat of 330 s 21 and a K M for dehydroshikimate of 200 mM (Kubota et al, 2013). For the AroE enzyme from P. putida KT2440, we previously measured a k cat of 300 s 21 and a K M for shikimate of 200 mM, albeit for the enzyme's reverse reaction (shikimate to dehydroshikimate) (Peek et al, 2011).…”

Mycobacterium tuberculosis Shikimate Pathway Enzymes as Targets for the Rational Design of Anti-Tuberculosis Drugs

“…In most bacteria, SD is encoded by the aroE gene, like in E. coli, M. tuberculosis and Methanococus jannaschii [83][84][85][86], while QD is encoded by yidB in E. coli [83], qsuD in Corynebacterium glutamicum [87], QA-3 in Neurospora crassa [88] and qutB in A. nidulans [89]. In some fungi, such as A. nidulans, N. crassa and Saccharomyces cerevisiae and in the apicomplexan parasite Toxoplasma gondii, SD is part of the multifunctional AROM enzyme complex, while in some plants like Arabidopsis thaliana and Populus trichocarpa, the enzyme is found in a bifunctional enzyme complex, with the third step of the pathway DHQase [90].…”

Identification and expression analysis of a gene encoding a shikimate transporter of Corynebacterium glutamicum

“…For this purpose, a mutant strain deficient in a shikimate kinase gene (aroK), a shikimate dehydrogenase gene (qsuD) and shiA was constructed. In our previous reports, the growth of a qsuD-deficient mutant was completely suppressed in BT medium supplemented with 50 mM shikimate (Kubota et al, 2013;Teramoto et al, 2009). This indicates that the triple-deletion mutant is unable to utilize shikimate.…”

“…RNA concentrations were determined spectrophotometrically at OD 260 . A one-step real-time quantitative reverse transcription PCR (qRT-PCR) was performed with the Power SYBR Green PCR master mix (Applied Biosystems) with 20 ng template RNA by using the 7500 Fast real-time PCR system (Applied Biosystems) as described previously (Kubota et al, 2013). The relative abundances of the target mRNAs were quantified based on the cycle threshold (C t ) value, which is defined as the number of cycles required in order to obtain a fluorescence signal above the background level.…”