Structural Analysis of Saccharomyces cerevisiae Dihydroorotase Reveals Molecular Insights into the Tetramerization Mechanism

Abstract

Dihydroorotase (DHOase), a dimetalloenzyme containing a carbamylated lysine within the active site, is a member of the cyclic amidohydrolase family, which also includes allantoinase (ALLase), dihydropyrimidinase (DHPase), hydantoinase, and imidase. Unlike most known cyclic amidohydrolases, which are tetrameric, DHOase exists as a monomer or dimer. Here, we report and analyze two crystal structures of the eukaryotic Saccharomyces cerevisiae DHOase (ScDHOase) complexed with malate. The structures of different DHOases were also compared. An asymmetric unit of these crystals contained four crystallographically independent ScDHOase monomers. ScDHOase shares structural similarity with Escherichia coli DHOase (EcDHOase). Unlike EcDHOase, ScDHOase can form tetramers, both in the crystalline state and in solution. In addition, the subunit-interacting residues of ScDHOase for dimerization and tetramerization are significantly different from those of other DHOases. The tetramerization pattern of ScDHOase is also different from those of DHPase and ALLase. Based on sequence analysis and structural evidence, we identify two unique helices (α6 and α10) and a loop (loop 7) for tetramerization, and discuss why the residues for tetramerization in ScDHOase are not necessarily conserved among DHOases.

A Complexed Crystal Structure of a Single-Stranded DNA-Binding Protein with Quercetin and the Structural Basis of Flavonol Inhibition Specificity

“…The inhibitory effect of myricetin has been established in several helicases such as the SARS coronavirus helicase [ 55 , 56 ], the replicative DnaB helicase [ 57 , 58 , 59 ], the RSF1010 RepA helicase [ 60 ], and the PriA helicase [ 61 ]. Moreover, myricetin could also inhibit the activities of several cyclic amidohydrolases [ 62 ] such as the bacterial enzymes dihydropyrimidinase [ 63 , 64 ], dihydroorotase [ 65 , 66 , 67 , 68 , 69 ], and allantoinase [ 68 , 70 ]. Thus, myricetin may be a competent “dirty drug” (a multitarget drug) against ESKAPE pathogens [ 17 ] and has broad application prospects.…”

Anticancer and Antioxidant Activities of the Root Extract of the Carnivorous Pitcher Plant Sarracenia purpurea

“…In this study, we identified that S. purpurea-root-acetone could inhibit the enzymatic activity of huDHOase. DHOase is the third enzyme in the de novo biosynthesis pathway of pyrimidine nucleotides (Figure 7A) and is considered an attractive target for potential antimalarial, anticancer, and antipathogen chemotherapy [17,[39][40][41]43,[54][55][56][57][58][59][60][61]. This enzyme contains a binuclear metal center (Znα/Znβ) and a residue Asp1686 (Figure 7B) crucial for the catalysis [42,[62][63][64][65].…”

“…Dihydroorotase (DHOase) [17,[39][40][41] is the third enzyme in the de novo biosynthesis pathway for pyrimidine nucleotides [42] and an attractive target for potential anticancer chemotherapy [43,44]. When the urea cycle is dysregulated, nitrogen will be redirected and used by the multifunctional enzyme CAD (carbamoyl phosphate synthetase/aspartate transcarbamoylase/DHOase) to increase pyrimidine synthesis in cancer cells [45].…”

“…Dihydroorotase (DHOase) [17,[39][40][41] is the third enzym pathway for pyrimidine nucleotides [42] and an attractive t chemotherapy [43,44]. When the urea cycle is dysregulated…”

Crystal structure of Methanococcus jannaschii dihydroorotase

“…14 The active site residues His56, His58, His168, His227, and Asp302, that coordinate the two Zn ions (Figure 1A), are invariant among DHOase structures. 5,[11][12][13][14] KCX137 is invariant in bacterial type II, III, and human, 11 as well as active fungal 13 DHOases. The aspartate that coordinates the two Zn ions in bacterial type I, for example, BaDHOase, is invariant in this subtype.…”