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J. Biol. Chem., Vol. 275, Issue 29, 22196-22201, July 21, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/29/22196    most recent M910111199v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nyyssölä, A. Articles by Reinikainen, T. Search for Related Content PubMed PubMed Citation Articles by Nyyssölä, A. Articles by Reinikainen, T.

Extreme Halophiles Synthesize Betaine from Glycine by Methylation^*

Antti Nyyssölä, Janne Kerovuo^§, Pasi Kaukinen^¶, Niklas von Weymarn, and Tapani Reinikainen^§

From the ^§ Danisco Cultor Innovation, Kantvik, Sokeritehtaantie 20, FIN-02460 Kantvik, Finland

Received for publication, December 21, 1999, and in revised form, April 2, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Glycine betaine is a compatible solute, which is able to restore and maintain osmotic balance of living cells. It is synthesized and accumulated in response to abiotic stress. Betaine acts also as a methyl group donor and has a number of important applications including its use as a feed additive. The known biosynthetic pathways of betaine are universal and very well characterized. A number of enzymes catalyzing the two-step oxidation of choline to betaine have been isolated. In this work we have studied a novel betaine biosynthetic pathway in two phylogenically distant extreme halophiles, Actinopolyspora halophila and Ectothiorhodospira halochloris. We have identified a three-step series of methylation reactions from glycine to betaine, which is catalyzed by two methyltransferases, glycine sarcosine methyltransferase and sarcosine dimethylglycine methyltransferase, with partially overlapping substrate specificity. The methyltransferases from the two organisms show high sequence homology. E. halochloris methyltransferase genes were successfully expressed in Escherichia coli, and betaine accumulation and improved salt tolerance were demonstrated.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The ability to adapt to fluctuations in external osmolarity is fundamental to survival of organisms. Glycine betaine (N,N,N-trimethylglycine), defined as betaine in this article, is one of the most common osmolytes and is synthesized by number of bacteria, plants, and algae (1-6). Many halophilic microorganisms are capable of accumulating very high levels of betaine. Concentrations well above 1 mol/kg water have been reported (3, 7). Also many plants synthesize and accumulate betaine in response to drought or salinity (5, 8). In addition, correlation between cold tolerance and intracellular betaine concentration has been established (9-12).

Betaine is synthesized from choline by oxidation. A choline dehydrogenase has been shown to catalyze the two-step reaction of choline to betaine in many microbes (13-15). Alternatively, the oxidation of choline to betaine can be catalyzed by a choline oxidase (16, 17). In plants, the first oxidation step from choline to betaine aldehyde is catalyzed by a choline mono-oxygenase (18). The second oxidation step from betaine aldehyde to betaine is catalyzed by a betaine aldehyde dehydrogenase. A betaine aldehyde dehydrogenase has also been found in a number of microbes (13, 19, 20) and plants (18, 21, 22).

Of all aerobic heterotrophic eubacteria investigated, only Actinopolyspora halophila and a related isolate synthesize betaine de novo (23). In addition, halophilic archaebacterial methanogens (24, 25), halotolerant and halophilic cyanobacteria (3), and halotolerant or halophilic anoxygenic phototrophic bacteria (23) have been shown to synthesize betaine from simple carbon sources.

The biosynthetic pathways of de novo synthesis of betaine are poorly understood. NMR studies on archaebacterial methanogens have suggested that betaine might be synthesized from glycine by a series of methylation reactions (25, 26). However, no enzymes catalyzing the reactions have successfully been isolated. A similar pathway has also been suggested to exist in anaerobic photorophic sulfur bacterium, Ectothiorhodospira halochloris (4).

In this study, we describe the enzymology of betaine biosynthesis in E. halochloris and demonstrate that a similar biosynthetic pathway exists in A. halophila. In addition, osmotic tolerance was improved when the genes encoding this pathway were cloned and expressed in Escherichia coli.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Methyltransferase Activity Assay-- A modification of the previously reported glycine-N-methyltransferase assay (36) was used. It is based on selective detection and analysis of radioactive reaction products, N-monomethylglycine (sarcosine), N,N-dimethylglycine, and betaine, after a methyl group transfer from [methyl-¹⁴C]S-adenosylmethionine (AdoMet)¹ to substrate. 25 µl of 0.1 M substrate (glycine, sarcosine, or dimethylglycine), 25 µl of Reaction Buffer (560 mM Tris-HCl, pH 7.5, 4 mM 2-mercaptoethanol, 50 µM MgCl₂, 160 µM EDTA), 25 µl of 4 mM S-adenosyl-L-methionine (45 nCi of S-adenosyl-L-[methyl-¹⁴C]methionine), and 25 µl of enzyme sample were mixed together. The reaction mixture was incubated for 30 min at 37 °C, and the reaction was stopped by adding 75 µl of charcoal suspension (133 g/liter in 0.1 M acetic acid) and incubated for 10 min at 0 °C. After centrifugation for 10 min, 75 µl of the supernatant was removed for assay in a liquid scintillation counter (LS 6000 IC, Beckman, Fullerton, CA). The enzyme activity is calculated as micromoles of methyl groups transferred per min. Protein concentrations were determined using Bio-Rad protein assay reagent.

HPLC Analysis of Sarcosine, Dimethylglycine, and Betaine-- The reaction products were analyzed by HPLC using Aminex HPX-87C column as described earlier (37). In order to detect the radioactive products formed in the enzymatic reaction, 200-µl fractions were collected during the chromatographic run and analyzed by a liquid scintillation counter as described above. The products were identified using a standard containing 1 mM mixture of sarcosine, dimethylglycine, and betaine.

Purification of A. halophila Sarcosine Dimethylglycine Methyltransferase (SDMT)-- A. halophila ATCC 27976 was grown aerobically at 37 °C in complex medium (38) containing 18.5% (w/v) NaCl. Typically 1 g of harvested cells were suspended in 1.5 ml of disruption buffer (22% (w/v) sucrose, 27 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 50 mM Tris-HCl, pH 7.5) and disrupted with an MSE Soniprep 150 sonicator in 20-ml batches with maximum power. The suspension was sonicated for 10 min in 30-s pulses with 2-min cooling intervals. The cell debris was removed by centrifugation at 28,000 × g at 1 °C for 30 min. Adenosine-Sepharose affinity chromatography was used for the purification of the SDMT. The affinity column was prepared as follows. 1 g of 5'-AMP-Sepharose 4B (Amersham Pharmacia Biotech) was swollen and washed with water as described by the manufacturer. The gel was equilibrated with calf intestinal alkaline phosphatase buffer (Finnzymes, Espoo, Finland), and 100 units of calf intestinal alkaline phosphatase (Finnzymes) was added. The gel was incubated for 2 h at 37 °C with occasional shaking and washed with 20 mM Tris-HCl, pH 7.5.

18 ml of A. halophila cell-free extract was applied to the affinity column (10 × 90 mm). The column was washed with 20 mM Tris-HCl, pH 7.5, until the absorbance at 280 nm became constant. The protein bound to the column was eluted with 1 mM S-adenosylmethionine in 20 mM Tris-HCl, pH 7.5. The active fractions were pooled and concentrated by ultrafiltration (Centriplus 30, Amicon, Beverly, MA).

Purification of E. halochloris Glycine Sarcosine Methyltransferase (GSMT)-- E. halochloris ATCC 35916 was grown as described previously (39). Typically 1 g of harvested cells was suspended in 1.5 ml of Reaction Buffer supplemented with 1 mM PMSF and 1 mM dithiothreitol. The cells were disrupted with an MSE Soniprep 150 sonicator in 5-ml batches with maximum power. The suspension was sonicated for 3 min in 15-s pulses with 2-min cooling intervals. The cell debris was removed by centrifugation at 28,000 × g at 1 °C for 30 min.

Ammonium sulfate was added to 25 ml of cell-free extract to achieve 20% saturation and incubated for 30 min at 0 °C. The solution was centrifuged at 15,000 × g, and the supernatant was applied to a butyl-Sepharose 4 FF (Amersham Pharmacia Biotech) (10 × 50 mm) column pre-equilibrated with 20% saturated ammonium sulfate in 20 mM Tris-HCl, pH 7.5. The column was washed with 45 ml of equilibration buffer and eluted with linear gradient of 20 to 0% ammonium sulfate (80 ml). The flow rate was 2 ml/min. The active fractions were pooled, and ammonium sulfate was removed by gel filtration (Sephadex G-25, Amersham Pharmacia Biotech). The sample was applied to a DEAE-Memsep 1000 HP (Millipore, Milford, MA) column pre-equilibrated with 20 mM Tris-HCl, pH 7.5. The column was washed with 15 ml of buffer and eluted with a linear NaCl gradient (0-1 M). The volume of the gradient was 60 ml, and the flow rate was 3 ml/min. The active fractions were pooled and concentrated by ultrafiltration (Centriplus 30, Amicon; Ultrafree MC 10,000 NMWL filter unit, Millipore). The concentrated sample (100 µl) was applied to a Superose 12-HR-30 (Amersham Pharmacia Biotech) column. 20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl was used as the elution buffer with flow rate of 0.4 ml/min. The fractions containing GSMT activity were collected and concentrated by ultrafiltration (Ultrafree MC 10,000 NWML filter unit, Millipore).

Characterization of the Purified Proteins-- The molecular weight was determined by analytical gel filtration with Superose 12-HR-30 (Amersham Pharmacia Biotech) column according to the instructions given by the manufacturer. SDS-gel electrophoresis was carried out using 12% polyacrylamide gels according to standard protocol (40). The N-terminal and tryptic peptides were purified and sequenced by using Perkin-Elmer/Applied Biosystems Procise 494A protein sequencing system as described previously (41).

Cloning of the Genes-- The genomic DNA from both microbes was isolated (42). The genomic DNAs were partially digested with SacI and ligated to SacI-digested dephosphorylated ZapII arms (Stratagene, La Jolla, CA) and packaged to  particles using Gigapack III Gold packing extract (Stratagene) according to protocol provided by manufacturer.

Degenerate primers were designed on the basis of N-terminal and tryptic peptide sequences of E. halochloris GSMT and A. halophila SDMT. Chromosomal DNA was used as the template DNA in the polymerase chain reactions. The polymerase chain reaction fragments were labeled with rediprime DNA labeling system (Amersham Pharmacia Biotech) according to the instructions given by the manufacturer and used as probes to screen genomic libraries. The positive  clones were cored and excised with ExAssist helper phage (Stratagene, San Diego, CA) to obtain phagemids. The obtained phagemids were used to transform E. coli SOLR' cells (Stratagene). The plasmid DNAs were CsCl gradient-purified (43) and sequenced.

Expression of the Cloned Genes in E. coli-- Primers homologous to the 5' and 3' ends of the genes were used to amplify the genes. NcoI and BglII restriction sites were added to the primer 5' and 3' ends (respectively) to facilitate cloning. Chromosomal DNA was used as the template DNA in the polymerase chain reactions. The amplified fragments were digested with NcoI and BglII and ligated into NcoI/BglII cut PQE-60 expression vectors (Qiagen, Santa Clara, CA). Competent XL-1 Blue MRF' cells carrying pREP4 plasmid (Qiagen) were transformed with this ligation mix (44).

Overnight cultures of the positive clones were diluted 1:4 into fresh LB broth supplemented with 100 µg/ml ampicillin and grown at 37 °C and 200 rpm for 30 min. Isopropyl--D-thiogalactopyranoside was added (1 mM final concentration), and the culture was grown for an additional 3 h. The cells from 20 ml of culture were separated by centrifugation (10 min, 1,000 × g) and suspended to 1 ml of Reaction Buffer supplemented with 1 mM PMSF and 1 mM dithiothreitol. The cells were disrupted with an MSE Soniprep 150 sonicator with maximum power. The suspension was sonicated for 1 min in 15-s pulses with 2-min cooling intervals. The cell debris was removed by centrifugation as described before.

Production of Betaine in E. coli-- Genes for E. halochloris GSMT and SDMT were cloned to pQE-60, and the resulting plasmid was transformed to competent E. coli XL-1 Blue MRF' cells carrying pREP4 plasmid (Qiagen) as described above. MM63 medium (45) containing 5 g/liter glucose and supplemented with 1.5 ml/liter of vitamin solution VA (46), 2 g/liter ampicillin, and 25 mg/liter kanamycin was used in the study. Concentrated overnight culture was used to inoculate 100 ml of medium without NaCl or with 0.3 M NaCl (final concentration). The culture was induced with 0.05 mM isopropyl--D-thiogalactopyranoside and grown at 37 °C and 200 rpm. The cells were harvested at early stationary phase by centrifugation at 1,500 × g for 5 min. The cell pellet was extracted with perchloric acid essentially as described previously (47). Betaine was determined from this extract by HPLC as described above. 2.5 ml of the culture was washed with NaCl solution containing the same concentration of NaCl as the corresponding cultivation, and the dry weight of the cells was determined. The mass fraction of NaCl of the dry weight was determined by titration with 0.05 M AgNO₃ and with 0.1% K₂CrO₄ as the indicator.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Demonstration of Betaine Biosynthesis in E. halochloris and A. halophila Cell Extracts-- The aim of the study was to characterize the biosynthetic pathway of betaine in two halophilic organisms, E. halochloris and A. halophila. In order to investigate the existence of the hypothesized glycine methylation pathway, methyltransferase activity was measured using AdoMet and glycine as substrates. The results clearly show that both E. halochloris and A. halophila cell extracts have methyltransferase activity on glycine, and AdoMet acts as the methyl group donor (Table I). Further analysis of the reaction products by HPLC (Fig. 1A) and activity measurement on sarcosine and dimethylglycine confirm that the organisms synthesize betaine from glycine in a three-step methylation reaction. Glycine is first methylated to sarcosine and then further to dimethylglycine and betaine.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   HPLC analysis of the methylation reaction products. The products of the enzymatic reactions were analyzed by HPLC as described under "Experimental Procedures." The retention times of the standards (sarcosine (S), dimethylglycine (D), and betaine (B)) are shown by triangles on the x axis. A, synthesis of methylation products in a reaction mixture containing A. halophila cell extract. 25 mM glycine (white circles), 25 mM sarcosine (black square), or 25 mM dimethylglycine (black circle) and 4 mM S-adenosyl-L-[methyl-14C]methionine were used as substrates. All methylation intermediates from glycine to betaine are detected. The relative amount of reaction products depends on the substrate used. Identical results were obtained with E. halochloris cell extract (data not shown). B, synthesis of methylation products in a reaction mixture containing 0.53 mg/ml purified E. halochloris GSMT and 0.7 mg/ml purified A. halophila SDMT. 1.25 mM glycine (white circle) or 20 mM glycine (black square) and 8 mM S-adenosyl-L-[methyl-14C]methionine were used as substrates. The relative amount of products is dependent on the concentration of glycine in the reaction mixture.

Isolation of the Methyltransferases-- In order to characterize the enzymology of the methylation pathway, the methyltransferases were purified from A. halophila and E. halochloris cell extracts. Fractionation of A. halophila cell extract on adenosine-Sepharose affinity column resulted in isolation of a 32-kDa polypeptide as judged by SDS-gel electrophoresis (data not shown). Molecular weight determination by gel filtration under non-denaturing conditions indicates that the protein is a monomer. The purified enzyme showed activity on sarcosine (0.36 µmol min¹ mg¹) and dimethylglycine (1.0 µmol min¹ mg¹). There was no activity on glycine. The enzyme was named according to the substrate specificity as SDMT.

Isolation of other methyltransferases from A. halophila cell extract was not successful. Despite attempts to stabilize the methyltransferase activity on glycine, activity was lost within 30 min after preparation of the cell extract. On the contrary, the enzymatic activity on glycine was found to be stable in the E. halochloris cell extract. After chromatographic purification, a 38-kDa protein was isolated. The enzyme had activity on glycine (0.52 µmol min¹ mg¹) and sarcosine (0.19 µmol min¹ mg¹). There was no activity on dimethylglycine. The enzyme was named GSMT.

Synthesis of Betaine in Vitro-- The enzyme activity data indicate that the three methylation reactions from glycine to betaine are catalyzed by two enzymes, which have partially overlapping substrate specificity. The hypothesis was further confirmed by the results from an in vitro synthesis experiment using purified enzymes. E. halochloris GSMT and A. halophila SDMT were incubated in a reaction mixture containing glycine and [methyl-¹⁴C ]AdoMet. The HPLC of the reaction mixture shows radioactive peaks corresponding to the retention times of sarcosine, dimethylglycine, and betaine (Fig. 1B). The ratio of the methylation products was found to be dependent on glycine concentration. A relatively higher amount of betaine was synthesized at low glycine concentration.

Cloning of the Methyltransferase Genes-- Positive clones with 3.5- (A. halophila) and 5.0-kilobase pair (E. halochloris) inserts were isolated, and the inserts were sequenced. The E. halochloris clone contained 2 ORFs coding for the methyltransferases, which were 807 and 840 base pairs in length. The A. halophila clone had only one ORF (1698 base pairs), which had significant sequence homology with the E. halochloris ORFs. Further sequence analysis and comparison to the peptide sequences of the purified proteins revealed that the first gene of E. halochloris clone codes for the GSMT (Fig. 2). A homologous sequence can be found in the N-terminal part of the A. halophila gene. The N-terminal amino acid sequence of the purified A. halophila SDMT can be found in the middle of the same ORF indicating that the C-terminal part of the gene encodes the SDMT. Thus in A. halophila the GSMT and SDMT are synthesized from a single gene, and SDMT is probably a proteolytic processing product. The E. halochloris SDMT is encoded by a separate gene. In addition to methyltransferases, both betaine operons contained a gene, which showed homology to a number of S-adenosylmethionine synthases (data not shown).

View larger version (54K): [in this window] [in a new window]   Fig. 2.   Homology comparison of the amino acid sequences of the GSMT and SDMT enzymes of E. halochloris and A. halophila. Identical amino acids are shown in blue and similar amino acids are shown in red. The N termini of the native proteins isolated from the cell extracts are underlined. The putative AdoMet-binding site consensus sequence (tttxhhDhGtGxGhh) can be found in the amino acid sequences of all proteins (t, polar or turn forming; h, hydrophobic; x, any amino acid).

Expression of the Methyltransferases in E. coli-- The functionality of the enzymes was further confirmed by overexpressing the isolated genes in E. coli under E. coli phage T5 promoter (Table II). The E. halochloris GSMT and SDMT were both expressed in active form. The substrate specificity of GSMT was identical to the native protein. In addition, the E. halochloris SDMT was shown to have similar substrate specificity as the A. halophila SDMT. The functionality of the A. halophila enzymes could be demonstrated only partially. When the A. halophila GSMT-SDMT gene was expressed in E. coli, only very low levels of SDMT activity could be detected. A protein corresponding to the size of GSMT-SDMT fusion was, however, found in the cell pellet (data not shown). Truncated A. halophila GSMT and SDMT were designed on the basis of sequence homology with E. halochloris genes. Whereas expression of the truncated GSMT was not successful in E. coli, truncated SDMT was successfully expressed in a soluble form. The soluble protein had activity on sarcosine and dimethylglycine and a low level of activity on glycine.

When E. halochloris GSMT and SDMT were overexpressed in E. coli, formation of intracellular betaine could be detected in minimal medium containing glucose as the sole carbon source. In medium containing 0.3 M NaCl the amount of betaine synthesized corresponded to 1% of the cell dry weight. The growth curves shown in Fig. 3 indicate that betaine synthesized inside the cells stimulates the growth of E. coli in high osmolarity.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   The growth curves of E. coli transformed with E. halochloris GSMT and SDMT genes. The cells were grown in MM63 medium containing 5 g/liter glucose as the sole carbon source. White circles indicate pQE-60 vector control, and black circles indicate pQE-60GSMT+SDMT. The cultivations were performed in duplicate. A, the transformant expressing the genes was capable of growing to a higher cell density in the presence of 0.3 M NaCl. In addition the apparent growth rate was higher (roughly 50%). The intracellular betaine concentration was 78 µmol/g cell dry weight. B, there was no significant difference in growth in medium lacking NaCl. The intracellular betaine concentration was 31 µmol/g cell dry weight. No betaine was detected in the control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A. halophila and E. halochloris are extreme halophiles capable of living in very high osmotic strength. They are also very efficient betaine producers. The intracellular betaine concentration of A. halophila may represent up to 33% of the cell dry weight.² E. halochloris has been shown to accumulate up to 2.5 M intracellular betaine concentration (7).

It has been reported earlier that E. halochloris does not have the known choline oxidation pathway to betaine (4). The data presented in this article support the hypothesis that E. halochloris synthesizes betaine from glycine by methylation. We have also shown that the methyltransferase pathway exists in a phylogenically distant extremophile, A. halophila. Indications of a similar pathway in halophilic methanogens have been reported (25, 26). Since these organisms are able to produce betaine directly from simple carbon sources, the methyltransferase pathway appears to be a universal route to betaine synthesis de novo. The methyltransferase pathway is effectively linked to the cell central metabolism. The precursor, glycine, is synthesized from 3-phosphoglycerate, which is an intermediate of glycolysis. This facilitates efficient channeling of the carbon flow to betaine. The methylation reaction uses AdoMet as the methyl group donor. The production of AdoMet is very costly to the cell. It has been calculated that a methyl group being reduced and activated to an active methyl group of AdoMet costs 12 ATP equivalents (27). In addition, AdoMet has a central role in cell metabolism and takes part in number of important methylation reactions involving synthesis of methionine and phosphatidylcholine or modification of amino acids and DNA, for example. Therefore the A. halophila and E. halochloris must have efficient mechanisms to maintain the balance of AdoMet metabolism.

Glycine N-methyltransferase catalyzing the first methylation step from glycine to sarcosine is known from mammalian cells, and it is well characterized (28, 29). Also the crystal structure is available (30). Despite the partially similar function, the properties of the mammalian enzyme are different. The substrate specificity of E. halochloris GSMT is broader, and it is able to catalyze the two-step reaction from glycine to dimethylglycine. Moreover, sequence comparison with known methyltransferases indicates that GSMT and SDMT belong to a previously unknown methyltransferase family. However, the consensus sequence for an AdoMet-binding site, as suggested by Bork and co-workers (31), can be found.

The methyltransferases of A. halophila and E. halochloris are very homologous. Despite the similarities, the two enzyme systems are different. E. halochloris enzymes are encoded by two separate genes, whereas a fusion protein is synthesized in A. halophila. Unfortunately the functionality of A. halophila GSMT could not be demonstrated. The problems encountered in the purification of the enzyme and insolubility of the GSMT-SDMT protein when produced in E. coli may indicate that the enzyme is membrane-bound. However, the two enzyme systems are functionally related. The E. halochloris GSMT was capable of synthesizing betaine in vitro when combined with SDMT isolated from A. halophila cell extract.

The betaine biosynthetic pathway of E. halochloris was successfully expressed in E. coli, and betaine was accumulated inside the cells in the presence of moderate osmolarity. In addition, the cells were capable of growing to a higher cell density. The results clearly indicate that the methyltransferase pathway can be used to improve the osmotic tolerance of heterologous organisms. Drought and soil salinity are among the most important factors limiting crop productivity. Although many plants synthesize betaine, there are several commercially important crops such as potato, rice, tomato, and tobacco, which do not accumulate betaine. The introduction of the choline oxidation pathway has been shown to increase salt and freezing tolerance of many plants (12, 32-35). Thus, it will be interesting to compare the efficiency of the methyltransferase pathway with the choline oxidation pathway for improving stress tolerance in plants.

	ACKNOWLEDGEMENTS

We thank Professor Matti Leisola, Dr. Heikki Ojamo, and Dr. Andrew Morgan for support, advice, and encouragement during this work.

	FOOTNOTES

^* This work was supported in part by the Finnish National Technology Agency (TEKES).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF216281, AF216282, and AF216283.

Currrent address: Helsinki University of Technology, Laboratory of Bioprocess Engineering, P. O. Box 6100, FIN-02015 HUT, Finland.

^¶ Current address: Haartman Institute, Dept. of Virology, P. O. Box 21, FIN-00014 University of Helsinki, Finland.

To whom correspondence should be addressed. Tel.: 358-9-2974619; Fax: 358-9-2982203; E-mail: Tapani.Reinikainen@danisco.com.

Published, JBC Papers in Press, May 1, 2000, DOI 10.1074/jbc.M910111199

² A. Nyyssölä, unpublished results.

	ABBREVIATIONS

The abbreviations used are: AdoMet, S-adenosylmethionine; HPLC, high performance liquid chromatography; PMSF, phenylmethylsulfonyl fluoride; SDMT, sarcosine dimethylglycine methyltransferase; GSMT, glycine sarcosine methyltransferase; ORF, open reading frame.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 275/29/22196    most recent M910111199v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nyyssölä, A. Articles by Reinikainen, T. Search for Related Content PubMed PubMed Citation Articles by Nyyssölä, A. Articles by Reinikainen, T.