Báo cáo khoa học: Role of the C-terminal extension in a bacterial tyrosinase Michael Fairhead and

Role of the C-terminal extension in a bacterial tyrosinase

Michael Fairhead and Linda Tho¨ ny-Meyer

EMPA, Swiss Federal Laboratories for Materials Testing and Research, Laboratory for Biomaterials, St Gallen, Switzerland

Introduction

Tyrosinases and the related catechol oxidases (collec￾tively termed polyphenol oxidases) comprise a family

of binuclear copper enzymes found in many species

of animals, plants, fungi and bacteria that use phe￾nol-like starting materials to produce a variety of

biologically important compounds, such as melanin

and other polyphenolic compounds [1–3]. These

type III copper proteins are capable of two activities:

monophenolase or cresolase activity (EC 1.14.18.1)

and diphenolase or catecholase activity (EC 1.10.3.1).

Both activities result in the formation of reactive

quinones, and these species are important intermedi￾ates in the biosynthesis of compounds such as

melanin.

Given the ability of tyrosinases to react with phenols

and its di-copper redox centres, they have been

proposed for use in a variety of biotechnological,

biosensor and biocatalysis applications [2]. One exam￾ple includes tyrosinase immobilization as an electro￾chemical biosensor for a range of phenolic compounds

[4]. The enzyme can also react with tyrosine found on

polypeptides, and the reactive quinones formed allow

for protein cross-linking to chitosan films as well as

protein-protein cross-linking [5,6].

The only available crystal structure of the tyrosin￾ases comes from the secreted enzyme of Streptomyces

castaneoglobisporus [7] tyrosinase. The structure shows

the enzyme in complex with its accessory caddie

protein (see below). The tyrosinase is predominately

a-helical in structure and contains six histidine residues

co-ordinating the two copper atoms that form the

active site of the enzyme. With respect to its overall

fold and active site architecture, the bacterial enzyme

is strongly similar to the related enzyme catechol

Keywords

C-terminal domain; melanin; tyrosinase;

Verrucomicrobium spinosum; zymogen

Correspondence

L. Tho¨ny-Meyer, EMPA, Swiss Federal

Laboratories for Materials Testing and

Research, Laboratory for Biomaterials,

Lerchenfeldstrasse 5, St Gallen, CH-9014,

Switzerland

Fax: +41 44 071 274 7788

Tel: +41 44 071 274 7792

E-mail: [email protected]

(Received 22 October 2009, revised

13 January 2010, accepted 22 February

2010)

doi:10.1111/j.1742-4658.2010.07621.x

The well studied bacterial tyrosinases from the Streptomyces sp. bacteria

are distinguishable from their eukaryotic counterparts by the absence of a

C-terminal extension. In the present study, we report that the tyrosinase

from the bacterium Verrucomicrobium spinosum also has such a C-terminal

extension, thus making it distinct from the Streptomyces enzymes. The

entire tyrosinase gene from V. spinosum codes for a 57 kDa protein (full￾length unprocessed form), which has a twin arginine translocase type signal

peptide, the two copper-binding motifs typical of the tyrosinase protein

family and the aforementioned C-terminal extension. We expressed various

mutants of the recombinant enzyme in Escherichia coli and found that

removal of the C-terminal extension by genetic engineering or limited tryp￾sin digest of the pro-form results in a more active enzyme (i.e. 30–100-fold

increase in monophenolase and diphenolase activities). Further studies also

revealed the importance of a phenylalanine residue in this C-terminal

domain. These results demonstrate that the V. spinosum tyrosinase is a new

example of this interesting family of enzymes. In addition, we show that

this enzyme can be readily overproduced and purified and that it will prove

useful in furthering the understanding of these enzymes, as well as their

biotechnological application.

Abbreviations

L-DOPA, L-3,4-dihydroxyphenylalanine; TAT, twin arginine translocase.

FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS 2083