Recombinant expression of N-terminal truncated mutants of the membrane bound mouse, rat and human flavoenzyme dihydroorotate dehydrogenase A versatile tool to rate inhibitor effects?

Abstract

Mammalian dihydroorotate dehydrogenase, the fourth enzyme of pyrimidine de novo synthesis is an integral protein of the inner mitochondrial membrane that faces the intermembrane space and is functionally connected to the respiratory chain via ubiquinone. Here, we describe the first cloning and analyzing of the complete cDNA of mouse dihydroorotate dehydrogenase. Based on our recent functional expression of the full-length rat and human dihydroorotate dehydrogenase, here we expressed N-terminal-truncated C-terminal-histidine-tagged constructs of the mouse, rat and human enzymes in Escherichia coli. These proteins were devoid of the N-terminal bipartite sequence consisting of the mitochondrial targeting sequence and adjacent hydrophobic domain necessary for import and proper location and fixation of the enzyme in the inner mitochondrial membrane. By employing metal-chelate affinity chromatography under native conditions, the enzymes were purified without detergents to a specific activity of more than 100 micromol x min(-1) x mg(-1) at pH optimum of 8.0--8.1. Flavin analyses by UV-visible spectrometry of the native enzymes gave fairly stoichiometric ratios of 0.6--1.2 mol flavin per mol protein. The kinetic constants of the truncated rat enzyme (K(m) = 11 microM dihydroorotate; K(m) = 7 microM ubiquinone) and human enzyme (K(m) = 10 microM dihydroorotate; K(m) = 14 microM ubiquinone) were very close to those recently reported for the full-size enzymes. The constants for the mouse enzyme, K(m) = 26 microM dihydroorotate and K(m) = 62 microM ubiquinone, were slightly elevated in comparison to those of the other species. The three truncated enzymes were tested for their efficacy with five inhibitors of topical clinical relevance against autoimmune disorders and tumors. Whereas the presence of the N-terminus of dihydroorotate dehydrogenase was essentially irrelevant for the efficacy of the malononitrilamides A77-1726, MNA715 and MNA279 with the rat and human enzyme, the N-termini were found to be important for the efficacy of the dianisidine derivative redoxal. Moreover, the complete N-terminal part of the human enzyme seemed to be of crucial importance for the 'slow-binding' features of the cinchoninic acid derivative brequinar, which was suggested to be one of the reasons for the narrow therapeutic window reported from clinical trials on its anti-proliferative and immunosuppressive action.

Functional Expression of Human Dihydroorotate Dehydrogenase (DHODH) in pyr4 Mutants of Ustilago maydis Allows Target Validation of DHODH Inhibitors In Vivo

“…The availability of several additional fungal genome sequences, in particular those of basidiomycetous origin, will surely clarify this issue. Interestingly, the heterolo- Our biochemical analysis of U. maydis DHODH revealed that the activity of the recombinant enzyme is significantly lower than that of mammalian DHODH (6 U/mg compared to 99 U/mg for the human enzyme) (55). The K m value of U. maydis DHODH is significantly higher (43 M for DHO) than that of human DHODH (9.7 M for DHO) (55).…”

“…Interestingly, the heterolo- Our biochemical analysis of U. maydis DHODH revealed that the activity of the recombinant enzyme is significantly lower than that of mammalian DHODH (6 U/mg compared to 99 U/mg for the human enzyme) (55). The K m value of U. maydis DHODH is significantly higher (43 M for DHO) than that of human DHODH (9.7 M for DHO) (55). In both cases, these values were determined for recombinant proteins lacking their N-terminal transmembrane domains.…”

Inhibition of Dengue Virus through Suppression of Host Pyrimidine Biosynthesis

“…4B, NITD-982 inhibited DHODH activity with a 50% inhibitory concentration (IC 50 ) of 103 nM. As a positive control, brequinar, a known DHODH inhibitor (46), exhibited an IC 50 of 2.1 nM.…”

Teriflunomide - A New Tableted Drug for Therapy of Remitting Disseminated Sclerosis (Review)

“…В ходе этих исследований были зафиксиро-ваны эмбриотоксические и тератогенные эффекты териф-луномида у крыс и кроликов. Стоит, однако, отметить, что терифлуномид ингибирует фермент ДГОДГ у крыс силь-нее, чем ДГОДГ человека [29], в результате чего его анти-пролиферативная активность у крыс в 145 раз больше [21]. Это может объяснить, почему схожая плазменная концентрация терифлуномида приводит к тератогенному эффекту у крыс, но не у человека.…”