Trypanosoma brucei is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of T. brucei.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• fosfofruktokinasy metabolismus   MeSH
• glukoneogeneze * genetika   MeSH
• glukosa metabolismus   MeSH
• glycerol metabolismus   MeSH
• lidé MeSH
• savci MeSH
• transaldolasa metabolismus   MeSH
• Trypanosoma brucei brucei * genetika   metabolismus   MeSH
• uhlík metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát MeSH
• fosfofruktokinasy MeSH
• glukosa MeSH
• glycerol MeSH
• transaldolasa MeSH
• uhlík MeSH

Moonlighting proteins have two different functions within a single polypeptide chain. Exploring moonlighting enzymes from the environment using the metagenomic approach is interesting. In the present study, a novel β-glucosidase gene, designated as bgl1D, with lipolytic activity (renamed Lip1C) was cloned through function-based screening of a metagenomic library from uncultured soil microorganisms. The deduced amino acid sequence comparison and phylogenetic analysis also indicated that Lip1C and other putative lipases are closely related. Biochemical characterization demonstrated that the maximum activity of the recombinant Lip1C protein occurs at pH 8.0 and 30°C using 4-nitrophenyl butyrate as substrate. The putative lipase had an apparent K(m) value of 0.88 mmol/L, a k(cat) value of 212/min, and a k(cat)/K(m) value of 241 L/mmol/min. Lip1C exhibited habitat-specific characteristics with 5 mmol/L AlCl(3), CuCl(2), and LiCl. The characterization of the biochemical properties of Lip1C enhances our understanding of this novel moonlighting enzyme isolated from a soil metagenome.

• MeSH
• Bacteria klasifikace   enzymologie   genetika   izolace a purifikace   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• beta-glukosidasa chemie   genetika   metabolismus   MeSH
• fylogeneze MeSH
• kinetika MeSH
• lipasa chemie   genetika   metabolismus   MeSH
• metagenom * MeSH
• molekulární sekvence - údaje MeSH
• půdní mikrobiologie * MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• stabilita enzymů MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• beta-glukosidasa MeSH
• lipasa MeSH