Petroselinum crispum, SDH (Mill.) SDH

Hýskova, Veronika, Belonozníkov, Katerina, Smeringaiova, Ingrida, Kavan, Daniel, Ingr, Marek & Ryslava, Helena, 2021, How is the activity of shikimate dehydrogenase from the root of Petroselinum crispum (parsley) regulated and which side reactions are catalyzed?, Phytochemistry (112881) 190, pp. 1-12 : 2-3

publication ID 10.1016/j.phytochem.2021.112881


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Petroselinum crispum


2.1. Characterization of P. crispum View in CoL View at ENA root SDH: typical high pH optimum and 61.5 kDa molecular weight

SDH activity was screened in crude extracts from 8 different vegetables. Onion ( Allium cepa L., bulb) and broccoli ( Brassica oleracea var. italica , florets) crude extracts had low SDH activity, whereas crude extracts from P. crispum root and zucchini ( Cucurbita pepo L. var. cylindrical , pulp) were identified as the richest sources of SDH among all plants tested. A high SDH activity per gram of fresh weight correlates with a low total phenolics content and vice versa ( Fig. A.1 View Fig ). SDH was purified by ion exchange and gel chromatography from P. crispum ( Petroselinum crispum ) root to a final specific activity of 470 ± 18 nmol. min -1.

SDH activity was pH-dependent, with a pH optimum between 7-8.5 and 9.5-10 in the physiological and reverse reactions, respectively ( Table 1 View Table 1 ). The molecular weight was 63 kDa when determined by red native electrophoresis ( Fig. A.2A View Fig ) and 60 kDa when assessed by gel chromatography ( Fig. A.2B View Fig ). The isoelectric point of P. crispum SDH was 4.5 ( Fig. A.2C View Fig ). Only one protein band with SDH activity was detected after native red electrophoresis and isoelectric focusing. During gel chromatography, SDH was eluted as a single peak. Thus, only one isoform of SDH is present in P. crispum root .

2.2. The kinetic properties of P. crispum SDH differ from those of other dehydrogenases

The kinetic parameters of the reaction catalyzed by P. crispum root SDH were studied in both directions, i.e., in the physiological (shikimate pathway) direction: NADPH + DHS → SA + NADP, and in the reverse direction: SA + NADP → NADPH + DHS ( Fig. 1 View Fig ). The Michaelis constants and maximal velocities are summarized in Table 1 View Table 1 . The maximal reaction rate for the reverse reaction from SA to DHS was 4.6-fold higher than in the shikimate pathway direction (DHS reduction). Because the saturating concentrations of NADP and SA were high, we also determined the apparent Michaelis constant at approximate conditions ( Table 1 View Table 1 ). Furthermore, excess substrate DHS inhibited the reaction ( Fig. A.3 View Fig ) with a substrate inhibition constant of KSS = 0.12 ± 0.07 mM ( Table 1 View Table 1 ).

2.3. Product inhibition analysis confirmed the ordered mechanism of the SDH-catalyzed reaction in both directions

The kinetic mechanism of the bisubstrate SDH reaction was analyzed in both directions. To identify the type of mechanism, we constructed Lineweaver-Burk diagnostical plots ( Fig. 1 View Fig ), a Hanes plot and an Eadie-Hofstee plot (data not shown) and performed product inhibition assays ( Fig. 2 View Fig , Table 2 View Table 2 ). All kinetic parameters including Vmax, Km, KA, Kic, Kiu, Ki, V* max, KSS, S 0.5 were calculated from non-linear regression using particular equations ( Bisswanger, 2002; Marangoni, 2003).

The initial rate of the reaction in the physiological direction was measured using several concentrations of DHS and NADPH, showing the typical Lineweaver-Burk plot of a sequential mechanism: straight lines with an intercept left to the ordinate ( Fig. 1 View Fig ) and KA 0.25 ± 0.13 mM. In this direction, the free enzyme binds to NADPH, which allows DHS, but not SA, binding in an ordered mechanism, thus partly explaining the mutual competition between NADPH and SA. NADPH binding apparently prevents NADP binding, and vice versa, leading to bilateral mutual competitive inhibition ( Fig. 3 View Fig , Table 2 View Table 2 ).

The direction of SA oxidation confirmed the strong affinity between the free enzyme and NADP. The mechanism was sequential but with a very low, almost immeasurable, dissociation constant for the complex enzyme – substrate (KA), thus it seems to look like a ping-pong. Obviously, the complex enzyme-NADP is very thermodynamically stable ( Fig. 1 View Fig ).

NADP binding enables both DHS and SA binding. The latter follows a classical ordered mechanism in the direction of SA + NADP → NADPH + DHS; in contrast, the former results in the formation of a dead-end complex. Its origin explains the competitive inhibition of DHS against the substrate SA. Thus, these findings further confirm the competition between NADP and NADPH ( Fig. 3 View Fig , Table 2 View Table 2 ).

DHS binding to the free enzyme prevents NADPH binding by forming a dead-end complex (enzyme-DHS) that precludes a disordered mechanism in the direction of NADPH + DHS → SA + NADP. In addition, another dead-end complex (enzyme-DHS-NADP) is also formed when NADP binds to the enzyme-DHS ( Fig. 3 View Fig ).

SA binding to the free enzyme prevents both NADP and NADPH binding also by forming a dead-end complex, which, in the case of NADP, prevents a disordered mechanism in the direction of SA + NADP → NADPH + DHS. In the case of NADPH, the formation of the dead-end complex completes the explanation for the competitive inhibition by the SA inhibitor against the NADPH substrate. Thus, SA competes with NADPH to bind to free enzyme, but not the other way around, that is, NADPH does not compete with SA since SA binding to the enzyme leads to a dead-end complex. Therefore, we proposed an ordered mechanism of the bi-substrate reaction catalyzed by SDH in both directions with three dead-end complexes (enzyme-DHS, enzyme-SA, and enzymeNADP-DHS) ( Fig. 3 View Fig ).













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