title: Leptospira interrogans encodes an ROK family glucokinase involved in a cryptic glucose utilization pathway.
abstract: Although Leptospira interrogans is unable to utilize glucose as its carbon/energy source, the LA_1437 gene of L. interrogans serovar Lai potentially encodes a group III glucokinase (GLK). The L. interrogans GLK (LiGLK) heterogeneously expressed in Escherichia coli was purified and proved to be a homodimeric enzyme with its specific activity of 12.3 ± 0.6 U/mg x protein determined under an improved assay condition (pH 9.0, 50 ° C), 7.5-fold higher than that assayed under the previously used condition (pH 7.3, 25 ° C). The improved sensitivity allowed us to detect this enzymatic activity of (5.0 ± 0.6) × 10(-3) U/mg x protein in the crude extract of L. interrogans serovar Lai cultured in standard Ellinghausen-McCullough-Johnson-Harris medium. The k(cat) and K(m) values for d-glucose and ATP were similar to those of other group III GLKs, although the K(m) value for ATP was slightly higher. Site-directed mutagenesis analysis targeting the conserved amino acid residues in the potential ATP-binding motif hinted that a proper array of Gly residues in the motif might be important for maintaining the conformation that was essential for its function. Gene expression profiling and quantitative proteomic data mining provided preliminary evidence for the absence of efficient systems involved in glucose transport and glycolysis that might account for the failure of glucose utilization in L. interrogans.
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhang
authors.forename: Yunyi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhong
authors.forename: Jun
authors.initials: J
authors.lastname: Ma
authors.forename: Nanqiu
authors.initials: N
authors.lastname: Peng
authors.forename: Xingjun
authors.initials: X
authors.lastname: Cao
authors.forename: Chen
authors.initials: C
authors.lastname: Yang
authors.forename: Rong
authors.initials: R
authors.lastname: Zeng
authors.forename: Xiaokui
authors.initials: X
authors.lastname: Guo
authors.forename: Guoping
authors.initials: G
authors.lastname: Zhao
mesh_terms: D001483:Base Sequence; D003470:Culture Media; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005941:Glucokinase / Q000235:genetics*; D005947:Glucose / Q000378:metabolism*; D007700:Kinetics; D007921:Leptospira interrogans / Q000145:classification / Q000201:enzymology / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics
chemical_list: D003470:Culture Media; D017931:DNA Primers; D011994:Recombinant Proteins; D005941:Glucokinase; D005947:Glucose