Characterization of a novel glucosamine-6-phosphate deaminase from a hyperthermophilic archaeon.

Department of Synthetic and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto, Japan.
Journal of Bacteriology (Impact Factor: 3.19). 11/2005; 187(20):7038-44. DOI: 10.1128/JB.187.20.7038-7044.2005
Source: PubMed

ABSTRACT A key step in amino sugar metabolism is the interconversion between fructose-6-phosphate (Fru6P) and glucosamine-6-phosphate (GlcN6P). This conversion is catalyzed in the catabolic and anabolic directions by GlcN6P deaminase and GlcN6P synthase, respectively, two enzymes that show no relationship with one another in terms of primary structure. In this study, we examined the catalytic properties and regulatory features of the glmD gene product (GlmD(Tk)) present within a chitin degradation gene cluster in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Although the protein GlmD(Tk) was predicted as a probable sugar isomerase related to the C-terminal sugar isomerase domain of GlcN6P synthase, the recombinant GlmD(Tk) clearly exhibited GlcN6P deaminase activity, generating Fru6P and ammonia from GlcN6P. This enzyme also catalyzed the reverse reaction, the ammonia-dependent amination/isomerization of Fru6P to GlcN6P, whereas no GlcN6P synthase activity dependent on glutamine was observed. Kinetic analyses clarified the preference of this enzyme for the deaminase reaction rather than the reverse one, consistent with the catabolic function of GlmD(Tk). In T. kodakaraensis cells, glmD(Tk) was polycistronically transcribed together with upstream genes encoding an ABC transporter and a downstream exo-beta-glucosaminidase gene (glmA(Tk)) within the gene cluster, and their expression was induced by the chitin degradation intermediate, diacetylchitobiose. The results presented here indicate that GlmD(Tk) is actually a GlcN6P deaminase functioning in the entry of chitin-derived monosaccharides to glycolysis in this hyperthermophile. This enzyme is the first example of an archaeal GlcN6P deaminase and is a structurally novel type distinct from any previously known GlcN6P deaminase.

  • [Show abstract] [Hide abstract]
    ABSTRACT: The technique is considered for the design of polarizer including a waveguide bend as the essential construction element. It is shown that a waveguide bend may be studied and designed as a polarizer.
  • [Show abstract] [Hide abstract]
    ABSTRACT: The objective of this study was to determine reasons behind the low oral (p.o.) bioavailability of glucosamine. By using male Sprague-Dawley rats, the movement of glucosamine through everted gut, the effect of dose and glucose, and inhibition of a glucose transporter (GLUT2) by quercetin were studied. Glucosamine pharmacokinetics and the effect of dosing, route of administration, food and antibiotic to eradicate gut microflora was also studied. Both in vitro and in vivo studies demonstrated linear absorption kinetics for glucosamine. Absorption from duodenum was the greatest. Glucose had no effect on the transport, whereas quercetin significantly reduced the extent of glucosamine transport. Intraperitoneal doses were completely absorbed, whereas p.o. doses demonstrated low bioavailability, indicating the gut as the site of presystemic loss. Food had no significant effect on glucosamine pharmacokinetics. Antibiotic treatment resulted in strong trends towards increased bioavailability with significant increase in fecal recovery. Incubation of glucosamine with faeces resulted in a significant loss. Glucosamine's low bioavailability is, at least in part, due to its dependence on a transport-facilitated absorption and presystemic loss brought about by the gut microflora.
    Journal of Pharmaceutical Sciences 04/2012; 101(7):2574-83. · 3.13 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: BACKGROUND: The catabolic pathways of N-acetyl-D-galactosamine (Aga) and D-galactosamine (Gam) in E. coli were proposed from bioinformatic analysis of the aga/gam regulon in E. coli K-12 and later from studies using E. coli C. Of the thirteen genes in this cluster, the roles of agaA, agaI, and agaS predicted to code for Aga-6-P-deacetylase, Gam-6-P deaminase/isomerase, and ketose-aldolase isomerase, respectively, have not been experimentally tested. Here we study their roles in Aga and Gam utilization in E. coli O157:H7 and in E. coli C. RESULTS: Knockout mutants in agaA, agaI, and agaS were constructed to test their roles in Aga and Gam utilization. Knockout mutants in the N-acetylglucosamine (GlcNAc) pathway genes nagA and nagB coding for GlcNAc-6-P deacetylase and glucosamine-6-P deaminase/isomerase, respectively, and double knockout mutants DeltaagaA DeltanagA and [increment]agaI [increment]nagB were also constructed to investigate if there is any interplay of these enzymes between the Aga/Gam and the GlcNAc pathways. It is shown that Aga utilization was unaffected in DeltaagaA mutants but DeltaagaA DeltanagA mutants were blocked in Aga and GlcNAc utilization. E. coli C DeltanagA could not grow on GlcNAc but could grow when the aga/gam regulon was constitutively expressed. Complementation of DeltaagaA DeltanagA mutants with either agaA or nagA resulted in growth on both Aga and GlcNAc. It was also found that [increment]agaI, [increment]nagB, and [increment]agaI [increment]nagB mutants were unaffected in utilization of Aga and Gam. Importantly, [increment]agaS mutants were blocked in Aga and Gam utilization. Expression analysis of relevant genes in these strains with different genetic backgrounds by real time RT-PCR supported these observations. CONCLUSIONS: Aga utilization was not affected in DeltaagaA mutants because nagA was expressed and substituted for agaA. Complementation of DeltaagaA DeltanagA mutants with either agaA or nagA also showed that both agaA and nagA can substitute for each other. The [increment]agaI, [increment]nagB, and [increment]agaI [increment]nagB mutants were not affected in Aga and Gam utilization indicating that neither agaI nor nagB is involved in the deamination and isomerization of Gam-6-P. We propose that agaS codes for Gam-6-P deaminase/isomerase in the Aga/Gam pathway.
    BMC Microbiology 05/2013; 13(1):94. · 2.98 Impact Factor


Available from