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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  • Crystal Structure of Agaricus bisporus

    Tyrosinase

  • 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).

  • RIJKSUNIVERSITEIT GRONINGEN

    Crystal structure of Agaricus bisporus tyrosinase

    Proefschrift

    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

    door

    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

  • .

    .

    .

    .

  • Contents

    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

  • . . . .

  • 1

    Chapter 1

    An introduction to mushroom Agaricus bisporus tyrosinase

    Wangsa T. Ismaya, Harry J. Wichers, Bauke W. Dijkstra

  • 2

  • 3

    1. Tyrosinase and melanin Tyrosinases (also called monophenol monooxygenases, EC 1.14.18.1) 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

  • 4

    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

  • 5

    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

    reservation.

    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 (