Functional and structural characterization of domain truncated violaxanthin de-epoxidase

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Functional and structural characterization of domain truncated violaxanthin de-epoxidase. / Hallin, Erik Ingmar; Guo, Kuo; Åkerlund, Hans Erik.

In: Physiologia Plantarum, Vol. 157, No. 4, 01.08.2016, p. 414-421.

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TY - JOUR

T1 - Functional and structural characterization of domain truncated violaxanthin de-epoxidase

AU - Hallin, Erik Ingmar

AU - Guo, Kuo

AU - Åkerlund, Hans Erik

PY - 2016/8/1

Y1 - 2016/8/1

N2 - Photosynthetic organisms need protection against excessive light. By using non-photochemical quenching, where the excess light is converted into heat, the organism can survive at higher light intensities. This process is partly initiated by the formation of zeaxanthin, which is achieved by the de-epoxidation of violaxanthin and antheraxanthin to zeaxanthin. This reaction is catalyzed by violaxanthin de-epoxidase (VDE). VDE consists of three domains of which the central lipocalin-like domain has been the most characterized. By truncating the domains surrounding the lipocalin-like domain, we show that VDE activity is possible without the C-terminal domain but not without the N-terminal domain. The N-terminal domain shows no VDE activity by itself but when separately expressed domains are mixed, VDE activity is possible. This shows that these domains can be folded separately and could therefore be studied separately. An increase of the hydrodynamic radius of wild-type VDE was observed when pH was lowered toward the pH required for activity, consistent with a pH-dependent oligomerization. The C-terminally truncated VDE did not show such an oligomerization, was relatively more active at higher pH but did not alter the KM for ascorbate. Circular dichroism measurements revealed the presence of α-helical structure in both the N- and C-terminal domains. By measuring the initial formation of the product, VDE was found to convert a large number of violaxanthin molecules to antheraxanthin before producing any zeaxanthin, favoring a model where violaxanthin is bound non-symmetrically in VDE.

AB - Photosynthetic organisms need protection against excessive light. By using non-photochemical quenching, where the excess light is converted into heat, the organism can survive at higher light intensities. This process is partly initiated by the formation of zeaxanthin, which is achieved by the de-epoxidation of violaxanthin and antheraxanthin to zeaxanthin. This reaction is catalyzed by violaxanthin de-epoxidase (VDE). VDE consists of three domains of which the central lipocalin-like domain has been the most characterized. By truncating the domains surrounding the lipocalin-like domain, we show that VDE activity is possible without the C-terminal domain but not without the N-terminal domain. The N-terminal domain shows no VDE activity by itself but when separately expressed domains are mixed, VDE activity is possible. This shows that these domains can be folded separately and could therefore be studied separately. An increase of the hydrodynamic radius of wild-type VDE was observed when pH was lowered toward the pH required for activity, consistent with a pH-dependent oligomerization. The C-terminally truncated VDE did not show such an oligomerization, was relatively more active at higher pH but did not alter the KM for ascorbate. Circular dichroism measurements revealed the presence of α-helical structure in both the N- and C-terminal domains. By measuring the initial formation of the product, VDE was found to convert a large number of violaxanthin molecules to antheraxanthin before producing any zeaxanthin, favoring a model where violaxanthin is bound non-symmetrically in VDE.

UR - http://www.scopus.com/inward/record.url?scp=84978817827&partnerID=8YFLogxK

U2 - 10.1111/ppl.12428

DO - 10.1111/ppl.12428

M3 - Article

C2 - 26864799

VL - 157

SP - 414

EP - 421

JO - Physiologia Plantarum

JF - Physiologia Plantarum

SN - 0031-9317

IS - 4

ER -