ORIGINAL ARTICLE Switching from blue to yellow: altering the spectral properties of a high redox potential laccase by directed evolution DIANA M. MATE 1 , EVA GARCIA-RUIZ 1 , SUSANA CAMARERO 2 , VLADIMIR V. SHUBIN 3 , MAGNUS FALK 4 , SERGEY SHLEEV 4 , ANTONIO O. BALLESTEROS 1 & MIGUEL ALCALDE 1 1 Department of Biocatalysis, Institute of Catalysis, CSIC, Madrid, Spain, 2 Centro de Investigaciones Biológicas, CSIC, Madrid, Spain, 3 Laboratory of chloroplast biochemistry, A.N. Back Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia, and 4 Biomedical Sciences Laboratory, Faculty of Health and Society, Malmö University, Malmö, Sweden Abstract During directed evolution to functionally express the high redox potential laccase from the PM1 basidiomycete in Saccharomyces cerevisiae, the characteristic maximum absorption at the T1 copper site (Abs 610 T1Cu) was quenched, switching the typical blue colour of the enzyme to yellow. To determine the molecular basis of this colour change, we characterized the original wild-type laccase and its evolved mutant. Peptide printing and MALDI-TOF analysis con- firmed the absence of contaminating protein traces that could mask the Abs 610 T1Cu, while conservation of the redox potential at the T1 site was demonstrated by spectroelectrochemical redox titrations. Both wild-type and evolved laccases were capable of oxidizing a broad range of substrates (ABTS, guaiacol, DMP, synapic acid) and they displayed similar catalytic efficiencies. The laccase mutant could only oxidize high redox potential dyes (Poly R-478, Reactive Black 5, Azure B) in the presence of exogenous mediators, indicating that the yellow enzyme behaves like a blue laccase. The main consequence of over-expressing the mutant laccase was the generation of a six-residue N-terminal acidic extension, which was associated with the failure of the STE13 protease in the Golgi compartment giving rise to alternative process- ing. Removal of the N-terminal tail had a negative effect on laccase stability, secretion and its kinetics, although the truncated mutant remained yellow. The results of CD spectra analysis suggested that polyproline helixes were formed during the directed evolution altering spectral properties. Moreover, introducing the A461T and S426N mutations in the T1 environment during the first cycles of laboratory evolution appeared to mediate the alterations to Abs 610 T1Cu by affecting its coordinating sphere. This laccase mutant is a valuable departure point for further protein engineering towards different fates. Keywords: Yellow laccase, blue laccase, directed evolution, α-factor prepro-leader, redox potential, T1 copper coordination sphere Abbreviations: Abs 610 T1Cu, (maximum absorption at the T1 copper site); ABTS, 2,2 ′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); CD, circular dichroism; DMP, 2,6-dimethoxyphenol; ER, endoplasmic reticulum; E°‘T1, redox potential at the T1 Cu site; HBT, 1-hydroxybenzotriazole; HTP, high-throughput; ICP-OES, Inductively coupled plasma-optical emission spectrometry; IVOE, in vivo overlap extension; MALDI-TOF, Matrix Assisted Laser Desorption and Ionization-Time Of Flight; MRT, mediated redox titration; MW, molecular weight; pI, isoelectric point; NHE, Normal Hydrogen Electrode; PAH, polycyclic aromatic hydrocarbon; PVDF, polyvinylidene difluoride; RB5, reactive black 5; T 50 , temperature at which the enzyme retains 50% of its activity after a 10 min incubation; ThL, Trametes hirsuta laccase. Correspondence: Miguel Alcalde, Department of Biocatalysis, Institute of Catalysis, CSIC, Marie Curie 2, 28049, Madrid, Spain. Tel: + 34 915854806. Fax: + 34 915854760. E-mail: malcalde@icp.csic.es (Received 29 May 2012; revised 9 July 2012; accepted 11 November 2012) Introduction Fungal laccases (benzenodiol: oxygen oxidoreductase, EC 1.10.3.2) are remarkable biocatalysts with a broad substrate specificity and very few requirements Biocatalysis and Biotransformation, 2013; 31(1): 8–21 ISSN 1024-2422 print/ISSN 1029-2446 online © 2013 Informa UK, Ltd. DOI: 10.3109/10242422.2012.749463 (Riva 2006; Rodgers et al. 2010). These enzymes can oxidize a wide range of compounds using oxygen from the air and releasing water as the sole by-product. Fungal laccases oxidize ortho- and Biocatal Biotransformation Downloaded from informahealthcare.com by Universidad Autonoma on 02/11/13 For personal use only.