Functional consequences of a gene duplication and fusion event in an arginine kinase

Abstract

Phosphagen kinases catalyze the reversible transfer of a high-energy phosphoryl group from phosphorylated guanidino storage compounds known as phosphagens to ADP in the following general reaction: phosphagen + MgADP + H + ↔ guanidino acceptor + MgATP. These reactions are typically found in cells that display high and variable rates of energy turnover, such as muscle fibers, neurons, spermatozoa and transport epithelia (Ellington, 2001). Phosphagen kinases comprise a discrete enzyme family consisting of creatine kinase (CK), arginine kinase (AK), glycocyamine kinase (GK), taurocyamine kinase (TK), hypotaurocyamine kinase (HTK), lombricine kinase (LK), opheline kinase (OK) and thalassamine kinase (ThK). CK and AK have been the most extensively studied. These reactions play a critical role in maintaining high values of the effective free energy change of ATP hydrolysis (∆GATP) during burst contractile activity in muscle (Newsholme et al., 1978;Meyer et al., 1984;Grieshaber et al., 1994;Ellington, 2001). To mediate this role, CK and AK activities in burst muscles are kept at very high levels so as to maintain the reaction near equilibrium with its substrates over a broad range of rates of ATP turnover (Meyer et al., 1984;Ellington, 2001).Sequence analyses of members of the phosphagen kinase family reveal a high degree of homology (at least 30-40% amino acid identity; Babbitt et al., 1986;Dumas and Camonis, 1993;Mühlebach et al., 1994;Suzuki and Furukohri, 1994), indicating a shared evolution from the same ancestral gene (Watts, 1971). AK has traditionally been regarded as being most closely related to the ancestral phosphagen kinase (Watts, 1971(Watts, , 1975 Arginine kinase (AK) from the foot of the razor clam Ensis directus consists of two full-length AK domains, denoted D1 and D2, fused in a single polypeptide chain. The full-length cDNA for Ensis AK was obtained and its deduced amino acid sequence was analyzed in the context of the X-ray crystal structure of a typical, monomeric AK. Both domains of Ensis AK contain most of the residues currently thought to be critical in catalysis, suggesting that both AK domains are catalytically competent. The fulllength Ensis AK, a D2-NusA-His-tag fusion protein and a D2-truncated AK (enterokinase cleavage product of the fusion protein) were expressed in Escherichia coli and purified. All recombinant AK constructs displayed high enzyme activity. Attempts at expressing active D1 alone, D2 alone or a D1-NusA-His-tag fusion protein were unsuccessful. The catalytic properties of the active proteins were compared with the corresponding properties of recombinant AK from the horseshoe crab Limulus polyphemus, which is a typical monomeric AK. In contrast to expectations, the kinetic results strongly suggest that Ensis AK has only one active domain, namely D2. The K cat values for all Ensis constructs were roughly twice that of typical AKs, indicating higher overall catalytic throughput at the competent active site. Furthermore, both the full-length and truncated D2 Ensis AKs showed n...

Two-domain arginine kinase from the deep-sea clam Calyptogena kaikoi—Evidence of two active domains

“…1. On the other hand, using Ensis two-domain AK Compaan and Ellington (2003) [WT] suggesting that the mutation affects not only the target position but also the overall three-dimensional structure of the enzyme, or that some domain-domain interaction has been generated when taking a two-domain structure. Similar subunit-subunit interaction has been reported for dimeric CK [Hornemann et al, 2000].…”

“…Subsequently, the same type of two-domain AKs was isolated in the heterodont clams Pseudocardium [Suzuki et al, 1998], Corbicula and Solen [Suzuki et al, 2002] as well as Ensis [Compaan and Ellington, 2003].…”

“…However, the failure to obtain a soluble domain 1 enzyme made this proposal uncertain. Compaan and Ellington (2003) expressed NusA-His tagged domain 1 and 2 of the two-domain Ensis AK, refolded its domain 1, and showed that the domain 1 has no AK activity. Thus, they concluded that only the domain 2 functions as AK.…”

The Substrate-free and -bound Crystal Structures of the Duplicated Taurocyamine Kinase from the Human Parasite Schistosoma mansoni

“…Guanidino kinases (EC 2.7.3), also called phosphagen kinases (PKs), 3 constitute a large family of enzymes that catalyze the Mg 2ϩ -dependent phosphoryl transfer from ATP to various guanidino substrates to yield ADP and an N-phosphorylated guanidino product termed phosphagen. Because PKs can also efficiently catalyze the reverse reaction, they allow the establishment of a phosphagen reservoir that is readily available for ATP regeneration and maintain the ATP/ADP balance in cells with high and fluctuating energy requirements (1).…”

“…Creatine kinases can be produced from isoform gene groups classified as cytoplasmic (CK), mitochondrial, or flagellar. Arginine kinases (AKs) are widely distributed in invertebrates and have been proposed to be the most closely related to a PK-like ancestor (3). Other PKs, such as taurocyamine kinase (TK), glycocyamine kinase (GK), or lombricine kinase, have been observed in annelids and allied groups (1,4).…”