Sporothrix schenckii: purification and partial biochemical characterization of glucosamine-6-phosphate synthase, a potential antifungal target.
The first committed step of the biosynthetic pathway leading to uridine-5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is catalyzed by glucosamine-6-phosphate synthase (GlcN-6-P synthase), an enzyme proposed as a potential antifungal chemotherapy target. Here, we describe the purification and biochemical characterization of the native enzyme from the dimorphic pathogenic fungus Sporothrix schenckii. The availability of the pure protein facilitated its biochemical characterization. The enzyme exhibited subunit and native molecular masses of 79 and 350+/-5 kDa, respectively, suggesting a homotetrameric structure. Isoelectric point was 6.26 and K(m) values for fructose-6-phosphate and L-glutamine were 1.12+/-0.3 and 2.2+/-0.7 mM, respectively. Inhibition of activity by UDP-GlcNAc was enhanced by Glc-6-P and phosphorylation stimulated GlcN-6-P synthase activity without affecting the enzyme sensitivity to the aminosugar. A glutamine analogue, FMDP [N(3)-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid] was a more potent inhibitor of activity than ADMP (2-Amino-2-deoxy-D-mannitol-6-phosphate) but the latter was a stronger inhibitor of growth in two culture media. To our knowledge, this is the first report on the purification and biochemical characterization of a non-recombinant GlcN-6-P synthase from a true dimorphic fungus. Inhibition of enzyme activity and fungal growth by specific inhibitors of GlcN-6-P synthase strongly reinforces the role of this enzyme as a potential target for antifungal chemotherapy.
Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Guanajuato, Gto, México.
Joaquín
J
González-Ibarra
Sławomir
S
Milewski
Julio C
JC
Villagómez-Castro
Carmen
C
Cano-Canchola
Everardo
E
López-Romero
D004791:Enzyme Inhibitors / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism; D005650:Fumarates / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007526:Isoelectric Point; D007700:Kinetics; D008358:Mannose / Q000031:analogs & derivatives / Q000378:metabolism; D008970:Molecular Weight; D011485:Protein Binding; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry; D013175:Sporothrix / Q000201:enzymology*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism; D015091:beta-Alanine / Q000031:analogs & derivatives / Q000378:metabolism
D004791:Enzyme Inhibitors; D005636:Fructosephosphates; D005650:Fumarates; D005656:Fungal Proteins; D021122:Protein Subunits; D005973:Glutamine; D015091:beta-Alanine; D014537:Uridine Diphosphate N-Acetylglucosamine; C027618:fructose-6-phosphate; C056276:N(3)-(4-methoxyfumaroyl)-2,3-diaminopropionic acid; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D008358:Mannose