Functional Characterization of Carbohydrate-Utilization Enzymes Encoded by the YCJ Gene Cluster, a Well Conserved Assembly of Genes Among Gram-Negative Bacteria in the Gut
Abstract
Scientists are slowly gaining a better understanding of the composition of the gut microbiota and the role it plays in maintaining metabolic homeostasis in the host. Metagenomic sequencing of various gut microbial species have revealed the correlation between the composition of the gut microbial population and a number of diseases. Although it is acknowledged that the gut microbiome encodes a number of enzymes that aid in the digestion of various dietary components, much is unknown about the underlying biochemical processes. The functional annotation of unknown proteins has not kept pace with the speed at which bacterial genomes from the gut are being sequenced. Sometimes the difficulty in determining functions of these uncharacterized proteins is compounded due to the misannotation of homologues present in various public databases. In this dissertation, efforts to elucidate the functions of enzymes in the ycj gene cluster in Escherichia coli K-12 are described. This gene cluster, which encodes for enzymes responsible for carbohydrate-utilization, is well conserved among a number of Gram-negative bacteria in the gut which implies that it plays an important role in degrading sugars.
The substrate profiles for six uncharacterized enzymes (YcjM, YcjQ, YcjR, YcjS, YcjT, and YcjU) of unknown function in Escherichia coli K-12 have been determined. YcjT was determined to be kojibiose phosphorylase which catalyzes the phosphorolysis of kojibiose into D-glucose and β-D-glucose-1-phosphate. This is the first such enzyme to be reported in E. coli. Kojibiose is a component of cell wall lipoteichoic acids in Gram-positive bacteria and is of interest as a potential low-calorie sweetener and prebiotic. YcjU was determined to be a β-phosphoglucomutase with activities against β-D-galactose-1-phosphate, β- D-mannose-1-phosphate, and β-D-allose-1-phosphate. These activities have not been reported previously in the other known homologues of this enzyme. YcjM catalyzes the phosphorolysis of α-(1,2)-D-glucose-D-glycerate. YcjS was determined to be a NAD-dependent D-glucose dehydrogenase, while YcjQ was found to oxidize D-gulose, making it the first reported dehydrogenase that utilizes this rare hexose. The products of the dehydrogenases are proposed to be substrates for YcjR, a putative epimerase. The structure of selenomethionine-derivatized YcjR was solved and binding of a potential substrate was studied after docking it into the active site.
Subject
gut microbiomeEscherichia coli
carbohydrate metabolism
artificial sweeteners
kojibiose
enzyme discovery
COG
ycj
Citation
Mukherjee, Keya (2018). Functional Characterization of Carbohydrate-Utilization Enzymes Encoded by the YCJ Gene Cluster, a Well Conserved Assembly of Genes Among Gram-Negative Bacteria in the Gut. Doctoral dissertation, Texas A & M University. Available electronically from https : / /hdl .handle .net /1969 .1 /174468.