Modification of substrate specificity and catalytic activity of haloalkane dehalogenase LinB by opening de novo access tunnels
P. Szelcsanyiova, A. Gora, J. Brezovsky, P. Dvorak, Z. Prokop, R. Chaloupkova, J. Damborsky
Loschmidt Laboratories, Department of Experimental Biology and Research
Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Brno,
358209@mail.muni.cz
Enzymes are natural catalysts accelerating
the rate of chemical reactions in living organisms. Some of the enzymes posses
the active site deeply buried inside the protein interior, which is connected
with surrounding environment by access pathways, called tunnels. The size,
shape and flexibility of the tunnels play a significant role in the entry of substrates and solvent into the enzyme active
site and egress of reaction products [1]. Haloalkane dehalogenase LinB from Sphingobium japonicum
UT26 is an enzyme that catalyzes hydrolytic
conversion of halogenated aliphatic hydrocarbons to
their corresponding alcohols and halide anions [2]. Its active site is located
in predominantly buried hydrophobic cavity that is linked
with the protein surface by several tunnels [3]. Several variants of haloalkane dehalogenases LinB with modified tunnels were rationally designed and
constructed by site directed and site saturation mutagenesis.
Constructed LinB variants with closed and re-opened de novo tunnels were expressed in Escherichia coli,
purified to homogeneity by metallo-affinity chromatography and
kinetically characterized. Substrate specificity of the enzymes was determined spectrophotometrically towards a set of thirty different halogenated substrates and compared with substrate
specificity profiles of the wild type dehalogenase enzymes.
Multivariate statistical analysis of collected data revealed significant
changes in substrate specificity profiles of tested enzymes. The LinB variants with closed tunnels formed a substrate specifity novel group, whereas the variants possessing de novo tunnels were classified into the
same substrate specificity group as the LinB wt. All variants
with closed tunnels showed considerably lower activities towards majority of tested
substrates compared to the LinB wt. On the contrary, LinB variants with de
novo tunnels showed comparable or even better activity than the wild type
enzyme. Rational engineering of protein tunnels thus represents powerful approach
for modification of substrate specificity and
catalytic activity of the enzymes with buried active sites.
1. Prokop et al.
(2012) Protein Engineering Handbook,
edited by S. Lutz, & U.T. Bornscheuer, pp.
421-464. Weinheim: Wiley-VCH.
2.
Nagata et al. (1997) Appl. Environ. Microbiol. 63: 3707-3710.
3.
Koudelakova et al. (2013) Biotechnol. J. 8: 32–45.