Structural and biochemical study on the dual coenzyme (NADP+/NAD+) specific isocitrate dehydrogenase from Trypanosoma brucei glycosome

概要

Isocitrate dehydrogenase (IDH) is a ubiquitous enzyme catalyzing the oxidative decarboxylation of isocitrate to α-ketoglutarate (αKG) with concomitant reduction of an electron acceptor. Depending on the type of electron acceptor and protein localization, IDHs are classified as cytosolic NADP+-IDH (type 1 IDH), mitochondrial NADP+-IDH (type 2 IDH) and mitochondrial NAD+-IDH (type 3 IDH). Type 1 and type 2 NADP+-IDHs are homologous isoforms, which are homodimeric proteins, producing NADPH as an important reducing equivalent in the process of fatty acid synthesis (in cytoplasm) and reactive oxygen species scavenging system (in mitochondria). Different from NADP+- IDHs, type 3 IDH is a heterotetrameric protein, producing NADH for mitochondrial energy transduction.

　Trypanosoma brucei (T. brucei) is a protozoan parasite that causes African sleeping sickness. The parasite is transmitted between mammalian hosts and the insect vector, tsetse fly. T. brucei cell differentiation together with series of biochemical and morphological changes plays an important role in the parasite’s transmission and survival in the changing environments of its host and vector. In its insect stage, the parasite depends on mitochondrial metabolism, where amino acids are the main nutrient source from tsetse fly. In the mammalian host bloodstream where glucose is abundant, the parasite mainly depends on energy produced by glycolytic pathway, which is mostly compartmentalized in a trypanosome-specific organelle, glycosome.

　This parasite conserves two NADP+-IDHs, localized in mitochondria (TbIDHm) and glycosomes (TbIDHg). TbIDHg is the first and only glycosomal IDH identified so far. Furthermore, it was recently found that mutant parasites deficient in TbIDHg were unable to differentiate inside the vector and could not colonize the salivary glands, providing genetic evidence of the first discovered metabolic enzyme involved in parasite’s differentiation. Despite of such evidence, the molecular and biochemical mechanisms played by TbIDHg in the differentiation process is still unclear. Therefore, in this thesis I report a comprehensive biochemical study and co-crystallographic analysis of TbIDHg.

　Amino acid sequence alignment shows a 65% identity between human type 1 IDH and TbIDHg, which implies that TbIDHg belongs to type 1 NADP+-IDH. This is consistent with its homodimeric structure determined by both size-exclusion chromatography and crystal structures. Although T. brucei lacks NAD+-IDH in its genome, the NAD+-dependent IDH activity was observed, which was abolished in parasite lacking TbIDHg, but not in TbIDHm-deficient mutant. Subsequent biochemical characterization using purified TbIDHg shows that different from other known IDHs, TbIDHg has dual specificity for the two coenzymes, capable to reduce both NADP+ and NAD+ at similar catalytic efficiency (Figure 1). The crystal structure of TbIDHg in complex with NADPH as well as NADH obtained in my study, provide the first structural basis of binding mechanism between NADH and a type 1 IDH active site. Site-directed mutagenesis studies of residues involved with substrate recognition and conformational change, upon binding of substrates, strengthen the results obtained from crystallographic studies.

　A gain-of-function have been previously reported for human NADP+-IDH with a single mutation at R132, which confers this enzyme the ability to reduce αKG using reducing equivalents from NADPH resulting in production of 2-hydroxyglutarate (2-HG) (Figure 1), a well described oncometabolite accumulated in various types of cancer cells. Interestingly, I show that purified wild type TbIDHg has an intrinsic ability to produce 2-HG which is enhanced by mutation at residue equivalent to R132 in human NADP+-IDH. The physiological role of 2-HG produced by T. brucei is still under investigation.

　The results obtained in my study provide novel biochemical and structural biology insights into the dual coenzyme specificity of TbIDHg. Because of the impermeability of glycosomal membrane to NADPH and NADH, my study give rise to new questions such as “why T. brucei needs a dual coenzyme specific IDH confined into the glycosomes?” and “which function is required for differentiation: production of NADH, NADPH or both?”. Future studies will be addressed to answer such important specific questions. Currently, I am conducting gene swapping experiments in order to replace TbIDHg by a single coenzyme specific IDH (NADP+ or NAD+ specific) targeted into the glycosomes and evaluate their ability to differentiate in the insect stage, which I expect to shed light on the physiological significance of this enzyme in T. brucei biology.