Isolation of the GFA1 gene encoding glucosamine-6-phosphate synthase of Sporothrix schenckii and its expression in Saccharomyces cerevisiae

Juan Francisco Sánchez-López; Joaquín González-Ibarra; Aurelio Álvarez-Vargas; Slawomir Milewski; Julio César Villagómez-Castro; Carmen Cano-Canchola; Everardo López-Romero

doi:10.1016/j.pep.2014.12.002

Isolation of the GFA1 gene encoding glucosamine-6-phosphate synthase of Sporothrix schenckii and its expression in Saccharomyces cerevisiae

Protein Expr Purif. 2015 Jun:110:57-64. doi: 10.1016/j.pep.2014.12.002. Epub 2014 Dec 13.

Authors

Juan Francisco Sánchez-López¹, Joaquín González-Ibarra¹, Aurelio Álvarez-Vargas¹, Slawomir Milewski², Julio César Villagómez-Castro¹, Carmen Cano-Canchola¹, Everardo López-Romero³

Affiliations

¹ Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, Mexico.
² Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdansk University of Technology, Gdansk, Poland.
³ Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, Mexico. Electronic address: everlope@ugto.mx.

PMID: 25514203
DOI: 10.1016/j.pep.2014.12.002

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.

Keywords: Antifungal target; Biochemical characterization; Purification; Recombinant glucosamine-6-phosphate synthase; Sporothrix schenckii.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cloning, Molecular
Enzyme Stability
Escherichia coli / genetics
Escherichia coli / metabolism
Fumarates / chemistry
Fungal Proteins / antagonists & inhibitors
Fungal Proteins / chemistry
Fungal Proteins / genetics
Fungal Proteins / isolation & purification*
Gene Expression
Genetic Complementation Test
Genomic Library
Glucosamine / analogs & derivatives
Glucosamine / chemistry*
Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / antagonists & inhibitors
Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / chemistry
Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / genetics
Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / isolation & purification*
Kinetics
Open Reading Frames
Plasmids / chemistry
Plasmids / metabolism*
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / isolation & purification
Saccharomyces cerevisiae / enzymology
Saccharomyces cerevisiae / genetics*
Sporothrix / chemistry*
Sporothrix / enzymology
beta-Alanine / analogs & derivatives
beta-Alanine / chemistry

Substances

Fumarates
Fungal Proteins
Recombinant Proteins
beta-Alanine
N(3)-(4-methoxyfumaroyl)-2,3-diaminopropionic acid
Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)
Glucosamine