On the Ancestral UDP-Glucose Pyrophosphorylase Activity of GalF from Escherichia coli.

Ana C Ebrecht,Miguel A Ballicora,Agnieszka M Orlof,Natalia Sasoni,Carlos M Figueroa,Alberto A Iglesias

doi:10.3389/fmicb.2015.01253

Abstract

In bacteria, UDP-glucose is a central intermediate in carbohydrate metabolism. The enzyme responsible for its synthesis is encoded by the galU gene and its deletion generates cells unable to ferment galactose. In some bacteria, there is a second gene, galF, encoding for a protein with high sequence identity to GalU. However, the role of GalF has been contradictory regarding its catalytic capability and not well understood. In this work we show that GalF derives from a catalytic (UDP-glucose pyrophosphorylase) ancestor, but its activity is very low compared to GalU. We demonstrated that GalF has some residual UDP-glucose pyrophosphorylase activity by in vitro and in vivo experiments in which the phenotype of a galU- strain was reverted by the over-expression of GalF and its mutant. To demonstrate its evolutionary path of “enzyme inactivation” we enhanced the catalysis by mutagenesis and showed the importance of the quaternary structure. This study provides important information to understand the structural and functional evolutionary origin of the protein GalF in enteric bacteria.

Highlights

The biosynthesis of glycoconjugates essentially depends on the availability of activated sugars. Leloir (1971) discovered the importance of nucleoside-diphosphate-sugars (NDP-sugars) in metabolism nearly 60 years ago after isolating UDP-glucose (UDP-Glc) from yeast
UDP-Glc has a number of important metabolic functions, including the biosynthesis of lypo- (Sandlin et al, 1995; Dean and Goldberg, 2002; Vilches et al, 2007) and exo-polysaccharides (Becker et al, 1998; Mollerach et al, 1998), capsule and membrane-derived oligosaccharides (Komeda et al, 1977; Sandlin et al, 1995; Bonofiglio et al, 2005), trehalose (Giaever et al, 1988; Boos et al, 1990; Padilla et al, 2004), and cellulose (Ross et al, 1991)
This value for GalF was four orders of magnitude lower than the observed for GalU. This should clarify some contradictory statements in the literature. This E. coli GalF protein was reported as inactive, but those assays were performed on crude extracts (Marolda and Valvano, 1996)

Summary

Introduction

The biosynthesis of glycoconjugates essentially depends on the availability of activated sugars. Leloir (1971) discovered the importance of nucleoside-diphosphate-sugars (NDP-sugars) in metabolism nearly 60 years ago after isolating UDP-glucose (UDP-Glc) from yeast. UDP-Glc has a number of important metabolic functions, including the biosynthesis of lypo- (Sandlin et al, 1995; Dean and Goldberg, 2002; Vilches et al, 2007) and exo-polysaccharides (Becker et al, 1998; Mollerach et al, 1998), capsule and membrane-derived oligosaccharides (Komeda et al, 1977; Sandlin et al, 1995; Bonofiglio et al, 2005), trehalose (Giaever et al, 1988; Boos et al, 1990; Padilla et al, 2004), and cellulose (Ross et al, 1991) The production of this key metabolite is mediated by UDP-Glc pyrophosphorylase (UDPGlc PPase, UTP:glucose-1-phosphate uridylyltransferase, EC 2.7.7.9). The UDP-Glc PPase is a key enzyme in Glc anabolism

Methods

Results

Conclusion