beta-Alanine betaine synthesis in the Plumbaginaceae. Purification and characterization of a trifunctional, S-adenosyl-L-methionine-dependent N-methyltransferase from Limonium latifolium leaves - PubMed (original) (raw)

beta-Alanine betaine synthesis in the Plumbaginaceae. Purification and characterization of a trifunctional, S-adenosyl-L-methionine-dependent N-methyltransferase from Limonium latifolium leaves

B Rathinasabapathi et al. Plant Physiol. 2001 Jul.

Abstract

beta-Alanine (beta-Ala) betaine is an osmoprotective compound accumulated by most members of the highly stress-tolerant family Plumbaginaceae. Its potential role in plant tolerance to salinity and hypoxia makes its synthetic pathway an interesting target for metabolic engineering. In the Plumbaginaceae, beta-Ala betaine is synthesized by S-adenosyl-L-methionine-dependent N-methylation of beta-Ala via N-methyl beta-Ala and N,N-dimethyl beta-Ala. It was not known how many N-methyltransferases (NMTases) participate in the three N-methylations of beta-Ala. An NMTase was purified about 1,890-fold, from Limonium latifolium leaves, using a protocol consisting of polyethylene glycol precipitation, heat treatment, anion-exchange chromatography, gel filtration, native polyacrylamide gel electrophoresis, and two substrate affinity chromatography steps. The purified NMTase was trifunctional, methylating beta-Ala, N-methyl beta-Ala, and N,N-dimethyl beta-Ala. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses indicated that the native NMTase is a dimer of 43-kD subunits. The NMTase had an apparent K(m) of 45 microM S-adenosyl-l-methionine and substrate inhibition was observed above 200 microM. The apparent K(m) values for the methyl acceptor substrates were 5.3, 5.7, and 5.9 mM for beta-Ala, N-methyl beta-Ala, and N,N-dimethyl beta-Ala, respectively. The NMTase had an isoelectric point of 5.15 and was reversibly inhibited by the thiol reagent p-hydroxymercuribenzoic acid.

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Figures

Figure 1

Figure 1

The synthetic pathway to β-Ala betaine. Each downward arrow represents an Ado-Met-dependent _N_-methylation step.

Figure 2

Figure 2

Anion-exchange chromatography using DEAE-fractogel. The procedure is described in “Materials and Methods.” NMTase activities (nmol h−1/fraction) against β-Ala (BA), _N_-methyl β-Ala (MM), and _N,N_-dimethyl β-Ala (DM) are indicated by squares, triangles, and stars, respectively. The predicted KCl gradient (20–300 m

m

) is shown in a dotted line. Protein content (mg/fraction), estimated by the modified Lowry's method (Peterson, 1977), is shown in white circles.

Figure 3

Figure 3

N,_N_-Dimethyl β-Ala substrate affinity column chromatography. Protein elution profile by OD280 (optical density at 280 nm) is shown for the unbound fraction (UB) and elutions (KW = 50 m

m

KCl wash, SE = substrate elution, and KE = 200 m

m

KCl elution). NMTase activities (nmol h−1/fraction) with β-Ala (BA), _N_-methyl β-Ala (MM), and _N,N_-dimethyl β-Ala (DM) measured in the wash and the elutions are shown in the inset.

Figure 4

Figure 4

Adenosine agarose affinity chromatography. See “Materials and Methods” for details. Protein elution profile by OD280 (optical density at 280 nm) of fractions is shown for unbound (UB), 200 m

m

KCl wash (KW), and substrate elution with 5 m

m

Ado-Met (AE). Note that absorbance in the Ado-Met elution is largely due to the Ado-Met and not protein. NMTase activities (nmol h−1/fraction) with β-Ala (BA), _N_-methyl β-Ala (MM), and _N,N_-dimethyl β-Ala (DM) measured in the unbound fraction; 200 m

m

KCl wash and Ado-Met elution are shown in the inset.

Figure 5

Figure 5

SDS-PAGE analysis of the purified L. latifolium NMTase and Photoaffinity labeling. Lane A, Precision SDS-protein markers (Bio-Rad 161-0362). Lane B, SDS-denatured protein (20 ng) from the adenosine agarose step (Table I), separated in a 12% (w/v) acrylamide gel, and stained with silver stain. Lane C, Partially purified (100-fold) NMTase fraction following photoaffinity labeling with _S_-Adenosyl-l-[_methyl_-3H]Met, SDS-PAGE, and autoradiography. Lane D, Partially purified (100-fold) NMTase fraction following photoaffinity labeling with _S_-Adenosyl-

l

-[_methyl_-3H]Met in the presence of _S_-adenosyl-

l

-homo-Cys (AdoHCy), SDS-PAGE, and autoradiography.

Figure 6

Figure 6

Kinetic analysis of L. latifolium NMTase protein. A, Effect of varying Ado-Met concentration on the reaction velocity shown in a plot of s/v versus s, where s is the substrate concentration and v is the velocity. Ado-Met concentration was varied from 0 to 300 μ

m

and β-Ala concentration was kept at 10 m

m

. Inset shows the direct plot. B, Effect of varying β-Ala on the reaction velocity shown in a plot of s/v versus s. β-Ala levels were varied between 0 and 10 m

m

. Ado-Met concentration was kept at 100 μ

m

.

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