Functional characterization of front-end desaturases from trypanosomatids depicts the first polyunsaturated fatty acid biosynthetic pathway from a parasitic protozoan.
A survey of the three kinetoplastid genome projects revealed the presence of three putative front-end desaturase genes in Leishmania major, one in Trypanosoma brucei and two highly identical ones (98%) in T. cruzi. The encoded gene products were tentatively annotated as Delta8, Delta5 and Delta6 desaturases for L. major, and Delta6 desaturase for both trypanosomes. After phylogenetic and structural analysis of the deduced proteins, we predicted that the putative Delta6 desaturases could have Delta4 desaturase activity, based mainly on the conserved HX(3)HH motif for the second histidine box, when compared with Delta4 desaturases from Thraustochytrium, Euglena gracilis and the microalga, Pavlova lutheri, which are more than 30% identical to the trypanosomatid enzymes. After cloning and expression in Saccharomyces cerevisiae, it was possible to functionally characterize each of the front-end desaturases present in L. major and T. brucei. Our prediction about the presence of Delta4 desaturase activity in the three kinetoplastids was corroborated. In the same way, Delta5 desaturase activity was confirmed to be present in L. major. Interestingly, the putative Delta8 desaturase turned out to be a functional Delta6 desaturase, being 35% and 31% identical to Rhizopus oryzae and Pythium irregulareDelta6 desaturases, respectively. Our results indicate that no conclusive predictions can be made about the function of this class of enzymes merely on the basis of sequence homology. Moreover, they indicate that a complete pathway for very-long-chain polyunsaturated fatty acid biosynthesis is functional in L. major using Delta6, Delta5 and Delta4 desaturases. In trypanosomes, only Delta4 desaturases are present. The putative algal origin of the pathway in kinetoplastids is discussed.
Tripodi
Karina E J
KE
Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Santa Fe, Argentina.
Buttigliero
Laura V
LV
Altabe
Silvia G
SG
Uttaro
Antonio D
AD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002849:Chromatography, Gas; D002850:Chromatography, Gel; D017931:DNA Primers; D019143:Evolution, Molecular; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000032:analysis / Q000096:biosynthesis*; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D014345:Trypanosoma / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D005231:Fatty Acids, Unsaturated; D044943:Fatty Acid Desaturases