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

Hong-Hsiang Guan,Chun-Jung Chen,Cheng-Yang Huang,En-Shyh Lin,Yen-Hua Huang

doi:10.3390/molecules26237249

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.

Highlights

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamoyl aspartate (CA-asp) to dihydroorotate (DHO) for the biosynthesis of pyrimidine nucleotides [1]. the de novo synthesis process of pyrimidines is conserved among all species, the gene products responsible for these enzyme activities differ (Figure 1)
carbamoyl phosphate synthetase (CPSase) and aspartate transcarbamoylase (ATCase) are present in a single bifunctional protein Ura2, which is a CAD-like polypeptide that contains a defective DHOase-like domain [3]
The DHOase activity was found in all organisms for the biosynthesis of pyrimidine nucleotides, but phylogenetic and structural analyses revealed at least three different

Summary

Introduction

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamoyl aspartate (CA-asp) to dihydroorotate (DHO) for the biosynthesis of pyrimidine nucleotides [1]. the de novo synthesis process of pyrimidines is conserved among all species, the gene products responsible for these enzyme activities differ (Figure 1). DHOase is a part of a single trifunctional polypeptide of 240 kDa, namely, carbamoyl phosphate synthetase (CPSase)/aspartate transcarbamoylase (ATCase)/DHOase protein (CAD), that it self-assembles into a hexamer of 1.5 MDa [2]. CPSase and ATCase are present in a single bifunctional protein Ura, which is a CAD-like polypeptide that contains a defective DHOase-like domain [3]. Given that CAD is a key enzyme for the cell’s progression through the S phase of the cell cycle and controls the cell proliferation in mammalian cells [6,7,8], these distinct differences among species may indicate that DHOase is a promising target [9,10,11] for potential antimalarial, anticancer, and antipathogen chemotherapy [12,13]

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