Identification of GutQ from Escherichia coli as a d -Arabinose 5-Phosphate Isomerase

Abstract

ABSTRACT The glucitol operon ( gutAEBDMRQ ) of Escherichia coli encodes a phosphoenolpyruvate:sugar phosphotransferase system that metabolizes the hexitol d -glucitol (sorbitol). The functions for all but the last gene, gutQ , have been previously assigned. The high sequence similarity between GutQ and KdsD, a d -arabinose 5-phosphate isomerase (API) from the 3-deoxy- d - manno -octulosonate (KDO)-lipopolysaccharide (LPS) biosynthetic pathway, suggested a putative activity, but its role within the context of the gut operon remained unclear. Accordingly, the enzyme was cloned, overexpressed, and characterized. Recombinant GutQ was shown to indeed be a second copy of API from the E. coli K-12 genome with biochemical properties similar to those of KdsD, catalyzing the reversible aldol-ketol isomerization between d -ribulose 5-phosphate (Ru5P) and d -arabinose 5-phosphate (A5P). Genomic disruptions of each API gene were constructed in E. coli K-12. TCM11[(Δ kdsD )] was capable of sustaining essential LPS synthesis at wild-type levels, indicating that GutQ functions as an API inside the cell. The gut operon remained inducible in TCM7[(Δ gutQ )], suggesting that GutQ is not directly involved in d -glucitol catabolism. The conditional mutant TCM15[(Δ gutQ Δ kdsD )] was dependent on exogenous A5P both for LPS synthesis/growth and for upregulation of the gut operon. The phenotype was suppressed by complementation in trans with a plasmid encoding a functional copy of GutQ or by increasing the amount of A5P in the medium. As there is no obvious obligatory role for GutQ in the metabolism of d -glucitol and there is no readily apparent link between d -glucitol metabolism and LPS biosynthesis, it is suggested that A5P is not only a building block for KDO biosynthesis but also may be a regulatory molecule involved in expression of the gut operon.