University of Groningen Crystal structure of Agaricus ... Agaricus bisporus tyrosinase Proefschrift
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University of Groningen
Crystal structure of Agaricus bisporus tyrosinase Ismaya, Wangsa Tirta
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Publication date: 2011
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Citation for published version (APA): Ismaya, W. T. (2011). Crystal structure of Agaricus bisporus tyrosinase. s.n.
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Crystal Structure of Agaricus bisporus
Cover: wall on the grass By: Wangsa Tirta Ismaya (2009)
The research was carried out at the Protein X-ray crystallography group, Laboratory of Biophysical Chemistry, Groningen Biomolecular Science and Biotechnology Institute, Faculty of Mathematics and Natural Sciences, University of Groningen, The Netherlands The research project was in collaboration with Agriculture and Food Innovation BV, Wageningen University and Research Center, and funded by Senter IOP (Industrial Onderzoek Project, IEE0002).
Crystal structure of Agaricus bisporus tyrosinase
ter verkrijging van het doctoraat in de Wiskunde en Natuurwetenschappen aan de Rijksuniversiteit Groningen
op gezag van de Rector Magnificus, dr. E. Sterken, in het openbaar te verdedigen op
vrijdag 24 juni 2011 om 14.45 uur
Wangsa Tirta Ismaya geboren op 12 januari 1975
te Jakarta, Indonesië
Promotores : Prof. dr. B. W. Dijkstra
Prof. dr. H. J. Wichers Beoordelingcommissie : Prof. dr. ir. M. W. Fraaije Prof. dr. E. Boekema Prof. dr. L. Dijkhuizen
ISBN : 978-90-367-4904-6 (electronic version) ISBN : 978-90-367-4905-3 (printed version)
For papi, mami, Lia, and Hery and all my teachers
and all my friends
Chapter 1 An introduction to mushroom Agaricus bisporus tyrosinase Page 1 – 23 Chapter 2 Overproduction, purification, and refolding of recombinant mushroom tyrosinase Page 25 – 40 Chapter 3 Crystallization and preliminary X-ray crystallographic analysis of mushroom Agaricus bisporus tyrosinase Page 41 – 52 Chapter 4 Crystal structure of mushroom Agaricus bisporus tyrosinase – identity of the tetramer and interaction with tropolone Page 53 – 74 References Page 75 – 81 Summary (English – Dutch – Indonesian) Page 83 – 99 Acknowledgement Page 101 – 102
. . . .
An introduction to mushroom Agaricus bisporus tyrosinase
Wangsa T. Ismaya, Harry J. Wichers, Bauke W. Dijkstra
1. Tyrosinase and melanin Tyrosinases (also called monophenol monooxygenases, EC 18.104.22.168) catalyze the o-
hydroxylation of phenols and the subsequent oxidation of the formed o-diphenols to
the corresponding o-quinones (Figure 1). Tyrosinase is the only copper-containing
enzyme known to perform these two subsequent reactions; the related catechol
oxidases only catalyze the second reaction, using o-diphenols as substrates
(Abolmaali, Taylor et al. 1998). Tyrosinases recruit molecular oxygen as a co-
substrate for the first reaction. One of the oxygen atoms is incorporated in the
phenolic ring while the other oxygen atom is reduced to a water molecule. This
catalytic cycle requires a reducing agent to provide two electrons to carry out the first
step. These two electrons originate from the second step of the reaction (Lind,
Siegbahn et al. 1999).
Figure 1. Reaction catalyzed by tyrosinase (Lind, Siegbahn et al. 1999).
Starting from tyrosine, the final product of the tyrosinase-catalyzed reaction is
dopaquinone. Dopaquinone can undergo subsequent chemical and enzymatic
reactions to result in either dopachrome or 5-cysteinyl dopaquinone derivatives,
which in turn can be further converted to melanin. The melanin biosynthesis via
dopachrome intermediate results in eumelanin, while the latter pathway results in
phaeomelanin and trichochrome (Figure 2). Melanins are pigments that can be found
in living organisms ranging from bacteria, plants, to mammals. Since tyrosinase
catalyzes the initial steps of this conversion, it is regarded as the key enzyme in the
melanin biosynthesis pathway (Sanchez-Ferrer, Rodriguez-Lopez et al. 1995).
o-Quinones and their derivatives such as melanin play important roles in the survival
of organisms. In mammals, melanin is mostly found in the skin, where it functions in
the photoprotection against UV radiation. In insects, o-quinones are components of
the protection system against pathogenic microorganisms and they are involved in the
hardening of cuticle, while in cephalopods o-quinones are the major components of
the defensive ink spray. Plants employ o-quinones in modifying and hardening the
protective exterior layer of, for instance, seed envelopes and as an agent against
invasive organisms. The latter function also occurs in fruits and potato, and in the
fruit bodies of fungi. These examples address the function of tyrosinase to be
associated with stress responses to the environment (Land, Ramsden et al. 2004).
Figure 2. Melanin biosynthesis pathway adapted from Sanchez-Ferrer et al. (1995).
2. Mushroom tyrosinase The first report on tyrosinase activity was the observation by Schönbein in 1856 of
the occurrence of aerobic oxidation in mushroom (Whitaker 1993). However,
attempts to isolate and purify mushroom tyrosinase were only started decades later
(Keilin and Mann 1938). To date, purification of the enzyme directly from mushroom
fruit bodies remains a difficult task because of contamination by endogenous
phenolic substrates and the presence of many tyrosinase isoforms (Jolivet, Arpin et
al. 1998). Even in recent years, partially purified enzyme is still employed for
biochemical studies (Gouzi and Benmansour 2007), which presents considerable
limitations as to the interpretation of findings (Flurkey, Cooksey et al. 2008).
Therefore, the use of commercially available mushroom tyrosinase, with known
enzyme activity, is often preferred.
Commercial mushroom tyrosinase is often employed in the search for inhibitors that
can be used for the treatment of skin disorders associated with melanin, or as
additives in cosmetics for skin whitening effects (Seo, Sharma et al. 2003; Chang
2009). It is also routinely employed in studying the application of the enzyme as a
food additive or as an element in detection systems of phenolic compounds in
wastewater (Solna, Sapelnikova et al. 2005; de Faria, Moure et al. 2007). The
commercial enzyme is however usually used without any auxiliary purification,
although it has been reported that impurities might have effects on enzyme activity
and inhibition (Rescigno, Zucca et al. 2007; Flurkey, Cooksey et al. 2008). The
unpurified commercial enzyme has even been used to study changes in secondary
structure content of the enzyme upon treatment with SDS (Karbassi, Haghbeen et al.
2003). The results of such latter studies should clearly be taken with considerable
2.1 Molecular characteristics of mushroom tyrosinase
2.1.1 Size and quaternary structure assembly
Detailed molecular studies require purified enzyme material. Starting from
commercially available mushroom powder, a heterotetrameric tyrosinase species with
a molecular mass of 120 kDa could be obtained after a single gel filtration
purification step (Schurink, van Berkel et al. 2007). This reported molecular mass of
the purified tyrosinase was in agreement with that reported by others, be it
determined by moving boundary electrophoresis (Mallette and Dawson 1949),
analytical centrifugation (