Functional characterization and stability improvement of a 'thermophilic-like' ene-reductase from Rhodococcus opacus 1CP.

Anika Riedel,Adrie H Westphal,Caroline E Paul,Willem J H Van Berkel,Marika Mehnert,Dirk Tischler

doi:10.3389/fmicb.2015.01073

Abstract

Ene-reductases (ERs) are widely applied for the asymmetric synthesis of relevant industrial chemicals. A novel ER OYERo2 was found within a set of 14 putative old yellow enzymes (OYEs) obtained by genome mining of the actinobacterium Rhodococcus opacus 1CP. Multiple sequence alignment suggested that the enzyme belongs to the group of ‘thermophilic-like’ OYEs. OYERo2 was produced in Escherichia coli and biochemically characterized. The enzyme is strongly NADPH dependent and uses non-covalently bound FMNH2 for the reduction of activated α,β-unsaturated alkenes. In the active form OYERo2 is a dimer. Optimal catalysis occurs at pH 7.3 and 37°C. OYERo2 showed highest specific activities (45-50 U mg-1) on maleimides, which are efficiently converted to the corresponding succinimides. The OYERo2-mediated reduction of prochiral alkenes afforded the (R)-products with excellent optical purity (ee > 99%). OYERo2 is not as thermo-resistant as related OYEs. Introduction of a characteristic intermolecular salt bridge by site-specific mutagenesis raised the half-life of enzyme inactivation at 32°C from 28 to 87 min and improved the tolerance toward organic co-solvents. The suitability of OYERo2 for application in industrial biocatalysis is discussed.

Highlights

Old Yellow Enzymes (OYE) or ene-reductases (ER) are flavoproteins catalyzing the asymmetric reduction of activated C = C bonds through a trans-hydrogenation reaction (Stürmer et al, 2007)
Sequence comparison with previously characterized ERs established that OYERo1 and OYERo3 belong to the ‘classical’ subclass while OYERo2 and OYERo4 group within the ‘thermophilic-like’ subclass (Figure 1)
OYERo2 drew our special attention since typically rhodococci do not encode for thermophilic proteins and that provided the motivation to investigate this enzyme

Summary

Introduction

Old Yellow Enzymes (OYE) or ene-reductases (ER) are flavoproteins catalyzing the asymmetric reduction of activated C = C bonds through a trans-hydrogenation reaction (Stürmer et al, 2007). Creating up to two stereogenic centers, OYEs are versatile biocatalysts for the biotransformation of cyclic and acyclic enones and enals, α,β-unsaturated dicarboxylic acids and esters, maleimides, terpenoids, nitroalkenes, steroids as well as nitrate esters and nitroaromatics (Leuenberger et al, 1976; Takabe et al, 1992; Vaz et al, 1995; Kataoka et al, 2002, 2004; Kurata et al, 2004; ChaparroRiggers et al, 2007; Hall et al, 2007; Stückler et al, 2007; Nivinskas et al, 2008; Fryszkowska et al, 2009; Adalbjörnsson et al, 2010; Mueller et al, 2010; Opperman et al, 2010; Toogood et al, 2010). Crystal structures of members of this group are determined for OYE1 (1OYA, 1OYB, and 1OYC; Fox and Karplus, 1994), PETNR (1H50; Barna et al, 2001), and morphinone reductase (1GWJ; Barna et al, 2002)

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Conclusion