TY - JOUR
T1 - Isolation of the GFA1 gene encoding glucosamine-6-phosphate synthase of Sporothrix schenckii and its expression in Saccharomyces cerevisiae
AU - Sánchez-López, Juan Francisco
AU - González-Ibarra, Joaquín
AU - Álvarez-Vargas, Aurelio
AU - Milewski, Slawomir
AU - Villagómez-Castro, Julio César
AU - Cano-Canchola, Carmen
AU - López-Romero, Everardo
N1 - Publisher Copyright:
© 2014 Elsevier Inc. All rights reserved.
PY - 2015/6
Y1 - 2015/6
N2 - 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 [N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme.
AB - 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 [N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme.
KW - Antifungal target
KW - Biochemical characterization
KW - Purification
KW - Recombinant glucosamine-6-phosphate synthase
KW - Sporothrix schenckii
UR - http://www.scopus.com/inward/record.url?scp=84923568994&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2014.12.002
DO - 10.1016/j.pep.2014.12.002
M3 - Artículo
C2 - 25514203
AN - SCOPUS:84923568994
SN - 1046-5928
VL - 110
SP - 57
EP - 64
JO - Protein Expression and Purification
JF - Protein Expression and Purification
ER -