Identification of two residues essential for the stringent substrate specificity and active site stability of the prokaryotic l‐arginine:glycine amidinotransferase CyrA

Abstract

A novel prokaryotic L-arginine:glycine amidinotransferase (CyrA; EC 2.1.4.1) is involved in the biosynthesis of the polyketide-derived cytotoxin cylindrospermopsin in the cyanobacterium Cylindrospermopsis raciborskii AWT250, and was previously characterized with regard to kinetic mechanism and substrate specificity [Muenchhoff J et al. (2010) FEBS J 277, 3844-3860]. In order to elucidate the structure-function-stability relationship of this enzyme, two residues in its active site were replaced with the residues that occur in the human L-arginine:glycine amidinotransferase (h-AGAT) at the corresponding positions (F245N and S247M), and a double variant carrying both substitutions was also created. In h-AGAT, both of these residues are critical for the function of this enzyme with regard to substrate binding, ligand-induced structural changes, and stability of the active site. In this study, we demonstrated that both single residue replacements resulted in a dramatic broadening of substrate specificity, but did not affect the kinetic mechanism. Experiments with substrate analogues indicate that donor substrates require a carboxylate group for binding. Evidence from initial velocity studies suggests that CyrA undergoes ligand-induced structural changes that involve Phe245. Stability parameters (T opt and T max ) of the CyrA variants differed from those of wild-type CyrA. Structural flexibilities of the wild type and all three variants were comparable on the basis of dynamic fluorescence quenching, indicating that changes in T opt are most likely attributable to localized effects within the active site. Overall, the results indicated that these two residues are essential for both stringent substrate specificity and the active site stability and flexibility of this unique cyanobacterial enzyme.

Characterization of Aphanizomenon ovalisporum amidinotransferase involved in cylindrospermopsin synthesis

“…2). Recently, it has been stated (Muenchhoff et al 2012) the role of these two residues in CyrA with the narrow substrate specificity previously observed by Muenchhoff et al(2010).…”

“…In general, AMDTs can use a wide variety of substrates. An exception is the AMDT from C. raciborskii AWT205, CyrA, the only cyanobacterial AMDT characterized to date (Muenchhoff et al 2010(Muenchhoff et al , 2012. In effect, CyrA, encoded by the cyrA gene, can use only L-arginine as a donor of the amidino group, and glycine as acceptor; therefore, it is considered an L-arginine:glycine AMDT (Muenchhoff et al 2010).…”

Cylindrospermopsin and Saxitoxin Synthetase Genes in Cylindrospermopsis raciborskii Strains from Brazilian Freshwater

“…Furthermore, these enzymes are involved in biosynthesis of the neurotoxin saxitoxin in cyanobacteria (SxtG, L–arginine/L–lysine amidinotransferase) and dinoflagellates [40], [62]. Despite a high level of conservation with regard to residues involved in catalysis and substrate binding, these enzymes have different substrate specificities and kinetic mechanisms [63]. The CyrA amidinotransferase was shown to be unique to the metabolic pathway for biosynthesis of CYN [12], [46].…”

Characterization of AmtA, an amidinotransferase involved in the biosynthesis of phaseolotoxins

“…It was also found that in hAGAT, Asn300 and Met302 play important roles in substrate binding and stability . To CyrA, substitution of Phe245 and/or Ser247 resulted in a broader substrate specificity .…”