Gene Cloning, Recombinant Expression, Characterization, and Molecular Modeling of the Glycolytic Enzyme Triosephosphate Isomerase from Fusarium oxysporum

Abstract

Triosephosphate isomerase (TPI) is a glycolysis enzyme, which catalyzes the reversible isomerization between dihydroxyactetone-3-phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP). In pathogenic organisms, TPI is essential to obtain the energy used to survive and infect. Fusarium oxisporum (Fox) is a fungus of biotechnological importance due to its pathogenicity in different organisms, that is why the relevance of also biochemically analyzing its TPI, being the first report of its kind in a Fusarium. Moreover, the kinetic characteristics or structural determinants related to its function remain unknown. Here, the Tpi gene from F. oxysporum was isolated, cloned, and overexpressed. The recombinant protein named FoxTPI was purified (97% purity) showing a molecular mass of 27 kDa, with optimal activity at pH 8.0 and and temperature of 37 °C. The values obtained for Km and Vmax using the substrate GAP were 0.47 ± 0.1 mM, and 5331 μmol min−1 mg−1, respectively. Furthemore, a protein structural modeling showed that FoxTPI has the classical topology of TPIs conserved in other organisms, including the catalytic residues conserved in the active site (Lys12, His94 and Glu164). Finally, when FoxTPI was analyzed with inhibitors, it was found that one of them inhibits its activity, which gives us the perspective of future studies and its potential use against this pathogen.

Bright Side of Fusarium oxysporum: Secondary Metabolites Bioactivities and Industrial Relevance in Biotechnology and Nanotechnology

“…Fusarium is a genus of filamentous fungi that includes many mycotoxin producers, agronomically important plant pathogens, and opportunistic human pathogens [12]. Its species are a widespread cosmopolitan group of fungi that are found in various habitats such as water, soil, or associated with plants [13,14]. They commonly colonize subterranean and aerial plant parts, either as primary or secondary invaders [15].…”

“…The Netherlands [56] Methoxydianthramide A methyl ester (14) 315 C 15 H 13 NO 5 F. oxysporum f. sp. dianthi Dianthus caryophyllus, stem (Caryophyllaceae)…”

Amoebicidal effect of 5,5′‐[(4‐nitrophenyl)methylene]bis‐6‐hydroxy‐2‐mercapto‐3‐methyl‐4(3H)‐pyrimidinone), a new drug against Entamoeba histolytica

“…[ 8–12 ] Also, some works have proposed a selective target, such as the enzyme triosephosphate isomerase (TIM) [ 13 ] ; this glycolytic enzyme has been used as a therapeutic target for the development of new drugs against various pathogenic organisms, such as Trypanosoma cruzi , Trypanosoma brucei , E. histolytica , Giardia duodenalis , Trichomonas vaginalis , Clostridium perfringens , among others. [ 14–24 ] In addition, other researchers are looking for the repositioning of commercial drugs as antiparasitics. [ 25,26 ] The triosephosphate isomerase of E. histolytica (EhTIM) [ 27 ] has an identity of 41.33% with human TIM (HsTIM); this difference in amino acids favors the development of new drugs with a selective target against EhTIM.…”

Potential Site to Direct Selective Compounds in the Triosephosphate Isomerase for the Development of New Drugs

“…In the pharmaceutical industry, the development of selective drugs to an enzyme or the repositioning of commercial drugs, today, is booming. The glycolytic enzyme triosephosphate isomerase (TIM) has been used as a therapeutic target for the development of new drugs against various pathogenic organisms, such as, Trypanosoma cruzi, Trypanosoma brucei, Entamoeba histolytica, Giardia duodenalis, Trichomonas vaginalis , among others . Therefore, saving resources in the development of new drugs, by directing the interaction of pharmacological compounds to a specific interaction site with a high probability to be selective, represents an opportunity, for researchers who are looking for new molecules or pharmacological compounds that they do not have a reported use, or in the case, of wanting to find another use to conventional drugs, such as omeprazole against TIM [11] [12] .…”

“…In this study, we propose a potential site as a therapeutic target against the enzyme triosephosphate isomerase for drug development, and that this potential new drug could be safe to be used at humans. We determined the K in position 214 (near the sequence YGGSV−K214) is indispensable for the interaction with the tested compounds and it helps the interaction in the active site from TIM.…”