Our previous research characterized two phosphoenolpyruvate (PEP) carboxylase (PEPC) isoforms (PEPC1 and PEPC2) from developing castor oil seeds (COS). The association of a shared 107-kD subunit (p107) with an immunologically unrelated bacterial PEPC-type 64-kD polypeptide (p64) leads to marked physical and kinetic differences between the PEPC1 p107 homotetramer and PEPC2 p107/p64 heterooctamer. Here, we describe the production of antiphosphorylation site-specific antibodies to the conserved p107 N-terminal serine-6 phosphorylation site. Immunoblotting established that the serine-6 of p107 is phosphorylated in COS PEPC1 and PEPC2. This phosphorylation was reversed in vitro following incubation of clarified COS extracts or purified PEPC1 or PEPC2 with mammalian protein phosphatase type 2A and is not involved in a potential PEPC1 and PEPC2 interconversion. Similar to other plant PEPCs examined to date, p107 phosphorylation increased PEPC1 activity at pH 7.3 by decreasing its K m (PEP) and sensitivity to L-malate inhibition, while enhancing glucose-6-P activation. By contrast, p107 phosphorylation increased PEPC2's K m (PEP) and sensitivity to malate, glutamic acid, and aspartic acid inhibition. Phosphorylation of p107 was promoted during COS development (coincident with a .5-fold increase in the I 50 [malate] value for total PEPC activity in desalted extracts) but disappeared during COS desiccation. The p107 of stage VII COS became fully dephosphorylated in planta 48 h following excision of COS pods or following 72 h of dark treatment of intact plants. The in vivo phosphorylation status of p107 appears to be modulated by photosynthate recently translocated from source leaves into developing COS.Phosphoenolpyruvate (PEP) carboxylase (PEPC; E.C. 4.1.1.31) is a ubiquitous and tightly regulated cytosolic enzyme in vascular plants that is also widely distributed in green algae and bacteria. PEPC catalyzes the irreversible b-carboxylation of PEP to yield oxaloacetate and inorganic phosphate (Pi). The enzyme plays a pivotal photosynthetic role in primary CO 2 fixation by C 4 and Crassulacean acid metabolism leaves Izui et al., 2004;Nimmo, 2005). PEPC also has a variety of additional important functions in plants, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates that are withdrawn for biosynthesis and nitrogen assimilation. Most native plant PEPCs are homotetramers, composed of identical subunits of approximately 100 to 110 kD, with crystal structures having been established for both the maize (Zea mays) and Escherichia coli enzymes (Izui et al., 2004). All PEPCs show allosteric properties: Vascular plant PEPCs are typically inhibited by L-malate and activated by Glc-6-P. In addition, Asp and Glu are allosteric inhibitors of PEPCs in plant tissues active in nitrogen assimilation and/or transamination reactions, thus providing a link between carbon and nitrogen metabolism (Law and Plaxton, 1997;Golombek et al., 1999;Moraes and Plaxton, 2000;Blonde and Plaxton, 2003). It is also well esta...

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 Δ8, Δ5 and Δ6 desaturases for L. major, and Δ6 desaturase for both trypanosomes. After phylogenetic and structural analysis of the deduced proteins, we predicted that the putative Δ6 desaturases could have Δ4 desaturase activity, based mainly on the conserved HX3HH motif for the second histidine box, when compared with Δ4 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 Δ4 desaturase activity in the three kinetoplastids was corroborated. In the same way, Δ5 desaturase activity was confirmed to be present in L. major. Interestingly, the putative Δ8 desaturase turned out to be a functional Δ6 desaturase, being 35% and 31% identical to Rhizopus oryzae and Pythium irregulareΔ6 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 Δ6, Δ5 and Δ4 desaturases. In trypanosomes, only Δ4 desaturases are present. The putative algal origin of the pathway in kinetoplastids is discussed.

Leishmania major synthesizes polyunsaturated fatty acids by using Δ6, Δ5 and Δ4 front‐end desaturases, which have recently been characterized [Tripodi KE, Buttigliero LV, Altabe SG & Uttaro AD (2006) FEBS J273, 271–280], and two predicted elongases specific for C18 Δ6 and C20 Δ5 polyunsaturated fatty acids, respectively. Trypanosoma brucei and Trypanosoma cruzi lack Δ6 and Δ5 desaturases but contain Δ4 desaturases, implying that trypanosomes use exogenous polyunsaturated fatty acids to produce C22 Δ4 fatty acids. In order to identify putative precursors of these C22 fatty acids and to completely describe the pathways for polyunsaturated fatty acid biosynthesis in trypanosomatids, we have performed a search in the three genomes and identified four different elongase genes in T. brucei, five in T. cruzi and 14 in L. major. After a phylogenetic analysis of the encoded proteins together with elongases from a variety of other organisms, we selected four candidate polyunsaturated fatty acid elongases. Leishmania major CAJ02037, T. brucei AAX69821 and T. cruzi XP_808770 share 57–52% identity, and group together with C20 Δ5 polyunsaturated fatty acid elongases from algae. The predicted activity was corroborated by functional characterization after expression in yeast. T. brucei elongase was also able to elongate Δ8 and Δ11 C20 polyunsaturated fatty acids. L. major CAJ08636, which shares 33% identity with Mortierella alpinaΔ6 elongase, showed a high specificity for C18 Δ6 polyunsaturated fatty acids. In all cases, a preference for n6 polyunsaturated fatty acids was observed. This indicates that L. major has, as predicted, Δ6 and Δ5 elongases and a complete pathway for polyunsaturated fatty acid biosynthesis. Trypanosomes contain only Δ5 elongases, which, together with Δ4 desaturases, allow them to use eicosapentaenoic acid and arachidonic acid, a precursor that is relatively abundant in the host, for C22 polyunsaturated fatty acid biosynthesis.