Isolation and Functional Characterization ofN-Methyltransferases That Catalyze Betaine Synthesis from Glycine in a Halotolerant Photosynthetic Organism Aphanothece halophytica

Abstract

Glycine betaine (N,N,N-trimethylglycine) is an important osmoprotectant and is synthesized in response to abiotic stresses. Although almost all known biosynthetic pathways of betaine are two-step oxidation of choline, here we isolated two N-methyltransferase genes from a halotolerant cyanobacterium Aphanothece halophytica. The most known biosynthetic pathways of betaine are the two-step oxidation of choline. Many bacteria, plants, and animals accumulate glycine betaine (here after betaine) under abiotic stress conditions (1-3). In these organisms, it was shown that betaine is synthesized by two steps, choline 3 betaine aldehyde 3 glycine betaine. The enzyme involved in the second step seems to be the same in plants, animals, and bacteria, namely NAD ϩ -dependent betaine-aldehyde dehydrogenase (4 -6). By contrast, different enzymes are involved for the first step. In plants, it was catalyzed by a novel Rieske-type iron-sulfur enzyme choline monooxygenase (7,8). In animals and many bacteria, the first step is catalyzed by membranebound choline dehydrogenase or soluble choline oxidase (9 -11). In some bacteria, choline dehydrogenase and choline oxidase also catalyze the second oxidation step (9 -11).It was suggested that betaine might be synthesized from glycine by a series of methylation reactions in archaebacterium Methanohalophilus portucalensis (12) and anaerobic phototrophic sulfur bacterium Ectothiorhodospira halochloris (13). Betaine synthesis from simple carbon sources has also been suggested in aerobic heterotrophic eubacterium Actinopolyspora halophila (13) and halotolerant cyanobacterium of Aphanothece halophytica (14). Recently, the methyltransferase genes that are involved in betaine synthesis have been isolated from E. halochloris and A. halophila (15). Two methyltransferase genes were involved in E. halochloris. One of gene products catalyzed the methylation reactions of glycine and sarcosine to sarcosine and dimethylglycine, respectively (EcGSMT), 1 whereas the other one catalyzed the methylations of sarcosine and dimethylglycine to dimethylglycine and betaine, respectively (EcSDMT) (15,16). By contrast, one ORF was found in A. halophila of which the N-and C-terminal parts had homologous sequences to those of EcGSMT and EcSDMT, respectively (15). The functionality of A. halophila methyltransferase was not well shown due to the formation of cell pellet when expressed in Escherichia coli.Glycine N-methyltransferase (GMT) catalyzing the methylation of glycine to sarcosine is known in mammalian cells although the enzymes catalyzing the further methylation steps do not occur (17,18). The homology of amino acid sequences between mammalian GMT and EcGSMT was low. No homologous sequences to those of EcGSMT, EcSDMT, and A. halophila methyltransferase could be found. Therefore, it was interesting to examine whether betaine is synthesized from glycine by three-step methylation reactions in other organisms.

Ecological Genomics of Marine Picocyanobacteria

“…In contrast, the marine cyanobacteria Trichodesmium erythraeum and, probably, Crocosphaera watsonii seem to harbor betAB. Recently, a stepwise methylation pathway was found to be responsible for GB synthesis in hypersaline cyanobacteria (311). Proteins similar to the corresponding methylating enzymes Gbmt1 (cluster 1941) and Gbmt2 (cluster 1942) from Aphanothece halophytica (Synechococcus sp.…”

Ectoine-Induced Proteins in Sinorhizobium meliloti Include an Ectoine ABC-Type Transporter Involved in Osmoprotection and Ectoine Catabolism

“…With the exceptions of proline and trehalose, the compatible solutes or osmoprotectants used by most bacteria are metabolically inert and their catabolism is unusual. A few phototrophic bacteria can synthesize GB de novo (45,65), but many bacteria can convert choline to GB in a two-step enzymatic oxidation reaction with glycine betaine aldehyde as the intermediate (6,12,40,54). Additionally, some bacteria can synthesize glycine betaine from carnitine (30,38,44).…”

Different strategies of osmoadaptation in the closely related marine myxobacteria Enhygromyxa salina SWB007 and Plesiocystis pacifica SIR-1

“…5a). It can be assumed that these genes are linked to betaine biosynthesis (Waditee et al, 2003). Downstream of these two methyltransferase genes, there is a gene putatively coding for the high-affinity betaine/carnitine/choline transporter, BetT.…”