Abstract
A 1332 bp full length cDNA encoding Teladorsagia circumcincta isocitrate lyase (TciICL) and a 1575 bp full length cDNA encoding T. circumcincta malate synthase (TciMS) were cloned, expressed in Escherichia coli and the recombinant proteins purified. The predicted TciICL protein of 444 amino acids was present as a single band of about 52 kDa on SDS-PAGE and the recombinant TciMS of 525 amino acids formed a single band about 62 kDa. Multiple alignments of the combined bifunctional TciICL-MS protein sequence with homologues from other nematodes showed that the greatest similarity (89–92 %) to the homologues of Ancylostoma ceylanicum, Haemonchus contortus and Haemonchus placei and 71–87 % similarity to the other nematode sequences. The 3-dimensional structures, binding and catalytic sites were determined for TciICL and TciMS and shown to be highly conserved. Substrate and metal ion binding sites were identified and were completely conserved in other homologues. TciICL was confirmed as a functional enzyme. At 30 °C, the optimum pH was pH 7.5, the Vmax was 275 ± 23 nmoles.min−1. mg-1 protein and the apparent Km for the substrate isocitrate was 0.7 ± 0.01μM (mean ± SEM, n = 3). Addition of 10 mM metal ions (except Mg2+) or 1 mM inhibitors reduced the recombinant TciICL activity by 60–90 %. Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TciICL in ELISA, supporting similar antigenicity to that of the native enzyme.
Highlights
The glyoxylate cycle (GC) converts isocitrate to malate in a two enzyme shunt which is an alternative to the full TCA cycle [1]
In the TCA cycle, isocitrate is the substrate for isocitrate dehydrogenase (ICD) and later malate is generated by fumarase; CO2 is generated at the steps catalysed by ICD and glutamate dehydrogenase (GDH)
The T. circumcincta GC enzymes isocitrate lyase (ICL) and malate synthase (MS) were sequenced separately, as typically the two nematode enzymes form a bifunctional protein, too large to be expressed in E. coli
Summary
The glyoxylate cycle (GC) converts isocitrate to malate in a two enzyme shunt which is an alternative to the full TCA cycle [1]. In the TCA cycle, isocitrate is the substrate for isocitrate dehydrogenase (ICD) and later malate is generated by fumarase; CO2 is generated at the steps catalysed by ICD and glutamate dehydrogenase (GDH). This shunt is active in bacteria, fungi, nematodes and plants, and the enzymes are reported to be inducible under specific conditions in other groups of animals under stress conditions [2, 3]. Enzyme activity has been measured in several freeliving species [4,5,6] and the animal parasitic Ascaris lumbricoides [7], Ascaris suum [8]
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