Characterization of four arginine kinases in the ciliate Paramecium tetraurelia: Investigation on the substrate inhibition mechanism

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Highlights

  • Four AKs from the ciliate Paramecium tetraurelia have been characterized.

  • AK3 showed substrate inhibition toward arginine. Residue S79 is essential for this unusual phenomenon.

  • The extremely low enzyme activity of AK4 was attributed to the unusual replacement of G298 by R.

Abstract

The ciliate Paramecium tetraurelia contains four arginine kinase genes (AK1-4). We detected cDNA for only three of the AKs (AK1–3) via PCR. Recombinant AK1–4 were expressed in Escherichia coli and their kinetics parameters determined. AK3 showed typical substrate inhibition toward arginine, and enzymatic activity markedly decreased when arginine concentration increased. This is the first example of substrate inhibition in wild-type phosphagen kinases. To explore the substrate inhibition mechanism, site-directed mutations were generated, targeting the amino acid sequence D-D-S-Q-V at positions 77–81 in P. tetraurelia AK3. Among the mutants, substrate inhibition was lost remarkably in the S79A mutant.

In spite of high amino acid sequence identity (91%) between P. tetraurelia AK3 and AK4, the enzymatic activity of AK4 was less by 3% than that of AK3. We noticed that the conservative G298 was unusually replaced by R in P. tetraurelia AK4, and we constructed two mutants, R298G/AK4 and G298R/AK3. Enzymatic activity of the former mutant was comparable with that of the wild-type AK3, whereas that of the latter mutant was dramatically reduced. Thus, we concluded that the significantly low activity of P. tetraurelia AK4 is due to the residue R298.

Graphical abstract

One of the Paramecium arginine kinases shows typical substrate inhibition toward arginine, and the Cys residue in C-terminal region of the enzyme is farnesylated. The residue S79 is responsible for the substrate inhibition.

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Introduction

Phosphagen kinases (PKs) are enzymes that catalyze the reversible transfer of the γ-phosphoryl group of ATP to guanidine substrates, yielding ADP and phosphorylated guanidine compounds referred to as phosphagens [1]. Although creatine kinase (CK) is the only PK in vertebrates, at least seven other enzymes are distributed in nonvertebrates: arginine kinase (AK), glycocyamine kinase, taurocyamine kinase (TK), hypotaurocyamine kinase, lombricine kinase, opheline kinase, and thalassemine kinase [2].

AK is widely distributed among invertebrates, such as arthropods, mollusks, and cnidaria [3] and is rarely found in unicellular organisms, such as bacteria [4], [5] and protozoan ciliates [3], [6]. TK activity has been found in protozoan oomycetes Phytophthora infestans and Phytophthora sojae [7], [8].

The ciliate Tetrahymena pyriformis has two AKs: a 40-kDa enzyme (AK1) and a 80-kDa enzyme consisting of a two-domain polypeptide chain (AK2) [9], [10]. T. pyriformis AK1 is localized in cilia [9], and the N-terminal glycine is myristoylated [11], suggesting that AK1 is anchored to the membrane via its myristoyl group. In a previous study, we proposed a phosphoarginine shuttle model in the cilia of T. pyriformis, in which the two AKs have distinct roles to transfer the high-energy phosphorylated group to the tip of cilia [9].

Tombes and Shapiro [12] was the first to propose the phosphagen (phosphocreatine) shuttle system in the flagellum of sea urchins, in which a myristoylated, unique three-domain CK is the key enzyme [13], [14]. Noguchi et al. [15] also proposed the phosphoarginine shuttle system in the cilia of Paramecium caudatum. Although they assumed that the AK enzyme plays an essential role in the model, the enzymes have not been identified and isolated. On the other hand, they estimated the effective concentration of AK is approximately 10 μM in the cilia of live P. caudatum [16]. A similar phosphoarginine shuttle system has also been proposed in the sperm flagella of the horseshoe crab Limulus polyphemus [17].

Prenylation (farnesylation or geranylgeranylation) is a type of posttranslational modification of proteins, which adds an isoprenyl group to the side chain of cysteines located near the C-terminal end [18], [19], [20]. The lipid moiety is involved in mediating protein and membrane contact. A prenylation signal sequence is mainly characterized by the following C-terminal four-amino-acid sequence: (C-[not E, D, N, and Q-any one of L, I, V, and M]-X). Farnesylation (C15) and geranylgeranylation (C20) are catalyzed by a farnesyltransferase and a geranylgeranyltransferase, respectively [21], [22]. Among PK sequences, the presence of the prenylation signal sequence is in the protozoan ciliate Paramecium tetraurelia AK and in the metazoan feather star Tropiometra afra macrodiscus AK [23].

The presence of four AK genes in P. tetraurelia was reported by Magida and Fraga [6]. P. tetraurelia AK genes have no intron, in contrast to those from T. pyriformis with three or four introns [5], [10]. In this paper, the DNA coding for the four P. tetraurelia AKs (AK1–4) were synthesized by overlap extension PCR, and their recombinant enzymes were expressed in E. coli. We found that AK3 demonstrated substrate inhibition toward arginine, and we elucidated that the S79 residue is responsible for this unusual phenomenon.

Section snippets

Isolation of total RNA from P. tetraurelia cells and cDNA synthesis

P. tetraurelia was grown at 25 °C in a hay infusion. P. tetraurelia cells (50 ml) were harvested by centrifugation at 890 × g for 15 min. Total RNA was isolated using ISOGEN II (Nippon Gene, Tokyo, Japan). Single-strand cDNA was synthesized by Ready-To-Go You-Prime First-Strand Beads (GE Healthcare-Amersham, NJ, USA) using a lock-docking oligo-dT primer with a BamHI site (5′- GGATCCT17V3N) [24].

PCR amplification of P. tetraurelia AK cDNA

The entire coding regions for P. tetraurelia AKs were amplified using pooled cDNA with the lock-docking

Detection of P. tetraurelia AK mRNA

Total RNA was isolated from P. tetraurelia cells to synthesize cDNA and assess expression of P. tetraurelia AKs. As shown in Fig. 1, cDNA for P. tetraurelia AK1, AK2, and AK3 were amplified. The products were subcloned, and sequences were confirmed to be identical with those deposited in the database. On the other hand, the cDNA for AK4 was not amplified (Fig. 1), suggesting that it is not expressed abundantly.

Comparison of amino acid sequences of P. tetraurelia AK1–4

P. tetraurelia AK1–4 consist of 388–392 amino acid residues with calculated molecular

Alignment of amino acid sequences of P. tetraurelia AK1–4 with other organismal AKs

The amino acid sequences of P. tetraurelia AK1–4 were aligned with the ciliate T. pyriformis AK1, the bacteria Sulfurovum lithotrophicum and Desulfotalea psychrophila AKs, and the horseshoe crab L. polyphemus AK in Fig. 3. The structure of the transition state analog complex (TSAC) of Limulus AK indicated that seven residues (S85, G86, V87, Y90, E254, C300, and E346: numbering in the alignment of Fig. 3) interact with the substrate arginine, and five residues (R145, R147, R257, R309, and R341:

Conclusion

Four AKs from the ciliate P. tetraurelia have been characterized. AK3 showed substrate inhibition toward arginine. This was the first example of substrate inhibition in the PK family. Residue S79 is essential for this unusual phenomenon. Interestingly, AK3 had a putative prenylation (farnesylation) signal sequence. The enzyme activity of AK4 was extremely low, which was attributed to the unusual replacement of G298 by R. The activity was recovered fully in the R298G mutant.

Acknowledgment

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan to TS (15K07151).

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