title: Isolation of the GFA1 gene encoding glucosamine-6-phosphate synthase of Sporothrix schenckii and its expression in Saccharomyces cerevisiae.
abstract: Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrix schenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases from other sources. The recombinant enzyme restored glucosamine prototrophy of the Saccharomyces cerevisiae gfa1 null mutant. Purification and biochemical analysis of the recombinant enzyme revealed some differences from the wild type enzyme, such as improved stability and less sensitivity to UDP-GlcNAc. The sensitivity of the recombinant enzyme to the selective inhibitor FMDP [N(3)-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme.
authors.affiliation: Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, Mexico.
authors.forename: Juan Francisco
authors.initials: JF
authors.lastname: Sánchez-López
authors.affiliation: Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, Mexico.
authors.forename: Joaquín
authors.initials: J
authors.lastname: González-Ibarra
authors.affiliation: Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, Mexico.
authors.forename: Aurelio
authors.initials: A
authors.lastname: Álvarez-Vargas
authors.affiliation: Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdansk University of Technology, Gdansk, Poland.
authors.forename: Slawomir
authors.initials: S
authors.lastname: Milewski
authors.affiliation: Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, Mexico.
authors.forename: Julio César
authors.initials: JC
authors.lastname: Villagómez-Castro
authors.affiliation: Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, Mexico.
authors.forename: Carmen
authors.initials: C
authors.lastname: Cano-Canchola
authors.affiliation: Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, Mexico. Electronic address: everlope@ugto.mx.
authors.forename: Everardo
authors.initials: E
authors.lastname: López-Romero
mesh_terms: D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005650:Fumarates / Q000737:chemistry; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression; D005816:Genetic Complementation Test; D015698:Genomic Library; D005944:Glucosamine / Q000031:analogs & derivatives / Q000737:chemistry*; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D016366:Open Reading Frames; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013175:Sporothrix / Q000737:chemistry* / Q000201:enzymology; D015091:beta-Alanine / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D005650:Fumarates; D005656:Fungal Proteins; D011994:Recombinant Proteins; D015091:beta-Alanine; C056276:N(3)-(4-methoxyfumaroyl)-2,3-diaminopropionic acid; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D005944:Glucosamine
keywords: Antifungal target; Biochemical characterization; Purification; Recombinant glucosamine-6-phosphate synthase; Sporothrix schenckii