Abstract
Hexokinase 2 (Hxk2p) from Saccharomyces cerevisiae is a bi-functional enzyme being both a catalyst and an important regulator in the glucose repression signal. In the presence of xylose Hxk2p is irreversibly inactivated through an autophosphorylation mechanism, affecting all functions. Consequently, the regulation of genes involved in sugar transport and fermentative metabolism is impaired. The aim of the study was to obtain new Hxk2p-variants, immune to the autophosphorylation, which potentially can restore the repressive capability closer to its nominal level. In this study we constructed the first condensed, rationally designed combinatorial library targeting the active-site in Hxk2p. We combined protein engineering and genetic engineering for efficient screening and identified a variant with Phe159 changed to tyrosine. This variant had 64% higher catalytic activity in the presence of xylose compared to the wild-type and is expected to be a key component for increasing the productivity of recombinant xylose-fermenting strains for bioethanol production from lignocellulosic feedstocks.
Highlights
The yeast Saccharomyces cerevisiae has three structural genes encoding enzymes that catalyse the phosphorylation of glucose to glucose 6-phosphate: HXK1, HXK2 and GLK1 [1]
The aim of the study was to identify a new variant of Hxk2p which i) is immune to autophosphorylation by xylose, ii) maintains its regulatory capability, and iii) has high catalytic activity in the presence of xylose
A homology model of Hxk2p was generated with a glucose moiety in the active site, based on the closed conformation of the S. cerevisiae Hxk1p scaffold
Summary
The yeast Saccharomyces cerevisiae has three structural genes encoding enzymes that catalyse the phosphorylation of glucose to glucose 6-phosphate: HXK1, HXK2 and GLK1 [1]. HXK1 and HXK2 encode hexokinases which, in addition to glucose, can phosphorylate fructose and mannose. These two proteins share a protein identity of 77%. GLK1 encodes a glucokinase which has an identity of 38% with the two hexokinases and lacks the ability to phosphorylate fructose [1]. During growth on fermentable carbon sources hexokinase 2 (Hxk2p) provides the main sugar-phosphorylating capability [2]. When the conditions change to nonfermentable carbon sources the HXK2 gene becomes repressed and the HXK1 and GLK1 genes are rapidly de-repressed [3]
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