Poster: Salinity

Abs # 152: Functional characterization of N-methyltransferases that syntheize betaine from glycine in a halotolerant cyanobacterium Aphanothece halophytica

Presenter:	Waditee, Rungaroon , waditee@ccmfs.meijo-u.ac.jp
Authors	Waditee, Rungaroon  (A)   Tanaka, Yoshito  (A)   Hibino, Takashi  (A)   Takabe, Tetsuko  (B)   Takano, Jun  (C)   Takabe, Teruhiro  (A)
Affiliations:	(A): Meijo University (B): Nagoya University (C): Shimadzu Company

Glycine betaine (N,N,N-trimethylglycine) is an important osmoprotectant and synthesized in response to abiotic stresses. Although almost all known biosynthetic pathways of betaine are two-step oxidation of choline, we isolated two N-methyltransferase genes from a halotolerant cyanobacterium Aphanothece halophytica. One of gene products (ORF1) catalyzed the methylation reactions of glycine and sarcosine with S-adenosylmethionine acting as the methyl donor. The other one (ORF2) specifically catalyzed the methylation of dimethylglycine to betaine. Both enzymes are active as monomers. Betaine, a final product, did not show the feed back inhibition for the methyltransferases even in the presence of 2 M. A reaction product, S-adenosyl homocysteine, inhibited the methylation reactions with relatively low affinities. The co-expressing of two enzymes in E. coli increased the betaine level and enhanced the growth rates. Immunoblot analysis revealed that the accumulation levels of both enzymes in Aphanothece halophytica cells increased with increasing the salinity. These results indicate that Aphanothece halophytica cells synthesize betaine from glycine by a three-step methylation. The changes of amino acids 169Arg to Lys or Glu in ORF1 and 171Pro to Gln and/or 172Met to Arg in ORF2 significantly decreased Vmax and increased Km for methyl acceptors (glycine, sarcosine, and dimethylglycine), but modestly affected Km for S-adenosylmethionine, indicating the importance of these amino acids for the binding of methyl acceptors. Physiological and functional properties of methyltransferases were discussed.