Crystal structure of Arabidopsis thaliana cytidine deaminase

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Highlights

  • Crystal structure of AtCDA1 is determined at 3.0 Å.

  • AtCDA1 is a dimeric zinc-containing enzyme.

  • Hydrogen bonds are the major force in the formation of AtCDA1 dimer.

  • The activities of AtCDA1 depend on the N-terminal catalytic deaminase domain.

  • The ribose moiety of cytidine is required for ligand binding.

Abstract

Cytidine deaminase (CDA) catalyzes the (deoxy)cytidine deamination to (deoxy)uridine, which involves in the catabolic and salvage pathways of pyrimidine nucleotides in plants. CDA serves as a prototype of the cytidine deaminase superfamily that contains a number of RNA editing enzymes. Arabidopsis thaliana has only one functional CDA, AtCDA1. We solved the crystal structures of AtCDA1, which is a dimeric zinc-containing enzyme and each protomer consists of an N-terminal zinc-binding catalytic domain and a C-terminal non-catalytic domain. Both domains adopt a typical α/β/α sandwich fold. In vitro biochemical assays showed that the ribose moiety of cytidine is required for ligand binding, and structural analyses revealed a conserved catalytic mechanism is adopted by AtCDA1.

Introduction

Cytidine deamination to uridine (C-to-U) or deoxycytidine deamination to deoxyuridine (dC-to-dU) is the first step of cytidine degradation and is part of the catabolic and salvage pathways of pyrimidine nucleotides in plants [1,2]. Recent studies indicate that the pyrimidine salvage pathway is crucial for chloroplast biogenesis [3]. C-to-U occurring at the mRNA and tRNA levels is a conserved RNA editing process that has been found in organisms from bacteria to mammals [4,5]. Enzymes catalyzing this process belong to the cytidine deaminase superfamily [6]. Its members include the classic cytidine deaminases (CDAs), apo-lipoprotein B mRNA editing catalytic complex (APOBEC) enzymes that are also named cytidine deaminases acting on RNA (CDARs), and those not specifically acting on (deoxy)cytidine, such as adenosine deaminases acting on RNA (ADARs) and adenosine deaminases acting on tRNA (ADATs). All these enzymes have a conserved zinc-containing catalytic domain, and the classic CDAs have been used as prototypes to understand the catalytic mechanism for this superfamily [7,8].

CDAs are evolutionarily conserved at the structural level, and there are two types of CDAs: homodimeric type (D-CDA) and homotetrameric type (T-CDA). D-CDA is about 32 kDa per protomer and its representative structure is the Escherichia coli CDA (EcCDA) [9,10]. T-CDA is about 15 kDa per protomer and represented by Bacillus subtilis CDA (BsCDA) and Homo sapiens (HsCDA) [11,12]. Both the N- or C-terminal domains of D-CDA show an α/β/α sandwich fold and only the N-terminal domain has the catalytic activity. In comparison, each protomer of the T-CDA has one active center and is half the size of a D-CDA protomer. The genome of Arabidopsis thaliana has nine CDA-like genes, but among them eight are pseudogenes and only CDA1 encodes an authentic enzyme (AtCDA1) that functions as the active cytidine deaminase in vivo [[13], [14], [15]]. In addition, the AtCDA1-catalyzed deamination can increase the number of G to A mutations during CaMV infection, which suggests an antiviral response similar to the APOBECs-mediated immunity in mammals [16].

Here, we report the crystal structure of AtCDA1, which belongs to D-CDA. The protomer consists of an N-terminal zinc-binding catalytic domain and a C-terminal non-catalytic domain. By in vitro biochemical experiments, we found that the catalytic activity of AtCDA1 requires the ribose moiety of cytidine. The reaction relies on the N-terminal domain and a catalytic mechanism conserved in the cytidine deaminase superfamily is employed.

Section snippets

Protein expression and purification

The full-length Arabidopsis CDA1 gene was amplified by PCR, introduced with a preceding tobacco etch virus (TEV) protease cleavage site and a following hexa-histidine tag, and then inserted into the pMAL-c5X vector (New England BioLabs, Ipswich, MA, USA) between Nde I and Sal I sites. The resulting vector was transformed into Escherichia coli BL21(DE3) cells, and the recombined protein was induced by 400 μM isopropyl β-D-thiogalactoside (IPTG) when OD600 reached 0.6–1.0 followed by additional

Overall structure

We crystallized the recombinant AtCDA1, the structure was solved and refined to 3.0 Å (Table 1). The atomic coordinates and structure factors (codes 6L08) have been deposited in the Protein Data Bank (http://wwpdb.org/). The overall AtCDA1 structure shows a dimeric arrangement and is similar to EcCDA (Fig. 1A). Each protomer consists of an N-terminal zinc-binding catalytic deaminase domain and a C-terminal non-catalytic deaminase domain. The total four domains form a pseudotetramer which is

Discussion

Cytidine deamination is also discovered as a mechanism of RNA editing, which underlies the genetic diversity and inhibition of retroviral infection, and appears to be vital to hosts’ heredity and immunity [31]. In flowering plants, the most frequent RNA editing events are C-to-U changes in mRNA, which occur frequently in chloroplasts and mitochondria and are affected by a GUN1-related multiple stress-related retrograde signaling [32]. The identified components of the RNA-editing complex include

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank Ming-Zhu Wang at Anhui University and the beamline scientists at the Shanghai Synchrotron Radiation Facility for technical support during data collection. This work was supported by the National Key R&D Program of China (2017YFA0503703), the China Postdoctoral Science Fund (2018M632517) and the Provincial Natural Science Foundation of Anhui (1908085QC129).

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