Characterization of Aphanizomenon ovalisporum amidinotransferase involved in cylindrospermopsin synthesis

Abstract

An increasing abundance of Aphanizomenon ovalisporum in water bodies from diverse world regions has been reported in the last few years, with the majority of the isolated strains producing the toxin cylindrospermopsin (CYN), leading to a rise in ecological and health risks. The understanding of CYN synthesis is crucial in the control of CYN production. An amidinotransferase (AMDT) seems to be the first enzyme involved in the synthesis of CYN. In this study, we have cloned and overexpressed the aoaA gene from the constitutive CYN producer A. ovalisporum UAM-MAO. The recombinant purified AoaA was characterized, confirming that it is an l-arginine:glycine AMDT. It shows an optimal activity between 32 and 37°C, at pH from 8 to 9. The activity exhibits a mixed (ping-pong/sequential) kinetic mechanism, and is inhibited by the reaction product guanidine acetate (GAA) in a noncompetitive manner. Mg2+ stimulates AoaA activity while Co2+ and Mn2+ inhibit it. AoaA conserves the critical residues of the catalytic site and substrate specificity of AMDTs, as the previously reported AMDT from Cylindrospermopsis raciborskii Cyr. Both proteins can be included in a new group of prokaryotic AMDTs involved in CYN production.

The virulence-associated protein HsvA from the fire blight pathogen Erwinia amylovora is a polyamine amidinotransferase

“…Kinetic parameters-With arginine as an amidino donor and putrescine as an acceptor, HsvA obeyed Michaelis-Menten kinetics with a K m value of 0.47 mM for arginine and 3.9 mM for putrescine (see Table 1). The K m value for putrescine is similar to values reported for acceptor substrates used in other amidinotransferases, including glycine in hAGAT, CyrA, and AoaA (9,14,15) and lysine in AmtA (19), with reported K m values ranging between 2.0 and 6.9 mM. Although the structurally related molecules 1,3-diaminopropane, cadaverine, and lysine all displayed low to moderate amidino acceptor activity under the conditions used in the substrate specificity profile screen, we were not able to measure K m values for these acceptors, as we observed only very low levels of product formation within the time frame of our kinetic assays at acceptor substrate concentrations Ͻ30 mM (data not shown), suggesting that these molecules are relatively inefficient substrates.…”

“…HsvA displayed maximum activity around a temperature of 35°C and a pH of 8. The temperature and pH activity profiles are similar overall to those reported for other bacterial amidinotransferases (14,15).…”

“…In prokaryotes, amidinotransferase enzymes have been found in the biosynthetic pathways of two aminoglycoside antibiotics, including streptomycin (11) and bluensomycin (12), and the antimycobacterial peptide antibiotics pheganomycin and resorcinomycin (13). Amidinotransferase enzymes are also known to participate in the biosynthetic pathways of several bacterial toxins, including cylindrospermopsin (14,15), saxitoxin (16), and phaseolotoxin (17,18). HsvA displays 48% amino acid identity with AmtA, the amidinotransferase found in the biosynthetic pathway for phaseolotoxin, a derivatized tripeptide phytotoxin produced in Pseudomonas syringae pv.…”

Dynamics of Cylindrospermopsin Production and Toxin Gene Expression in <i>Aphanizomenon ovalisporum</i>

“…The experiments were carried out with batch cultures of the strain of A. ovalisporum UAM-MAO [26], grown with nitrate as N source and under other conditions described by other authors as optima for A. ovalisporum. The conditions used seemed appropriate, as indicated by the maximum growth rate attained, 0.24 day -1 , similar to the value range previously reported, 0.2 -0.36 day −1 [2] [27].…”

Characterization of three amidinotransferases involved in the biosynthesis of ketomemicins

“…The best-studied amidinotransferase is L-arginine:glycine amidinotransferase in vertebrates, which is involved in creatine biosynthesis and plays an important role in the energy metabolism of muscle and nerve tissues. [4][5][6] Additionally, L-arginine:glycine amidinotransferases for cylindrospermopsin biosynthesis, 7,8 and an L-arginine:L-lysine amidinotransferase from Pseudomonas syringae 9 and an L-arginine:inosamine phosphate amidinotransferase from Streptomyces griseus for the biosynthesis of streptomycins 10 have been reported. In this article, we characterized three new amidinotransferases involved in the biosynthesis of ketomemicins.…”