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Identification and biochemical characterization of four wood-associated glucuronoxylan methyltransferases in Populus.

Yuan Y, Teng Q, Zhong R, Ye ZH - PLoS ONE (2014)

Bottom Line: The PtrGXM genes were found to be highly expressed in wood-forming cells and their encoded proteins were shown to be localized in the Golgi.Kinetic analysis showed that PtrGXMs exhibited differential affinities toward the GlcA-substituted xylooligomer acceptor with PtrGXM3 and PtrGXM4 having 10 times higher K m values than PtrGXM1 and PtrGXM2.Together, these findings indicate that PtrGXMs are methyltransferases mediating GlcA methylation in Populus xylan during wood formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
Wood is one of the promising bioenergy feedstocks for lignocellulosic biofuel production. Understanding how wood components are synthesized will help us design strategies for better utilization of wood for biofuel production. One of the major wood components is xylan, in which about 10% of xylosyl residues are substituted with glucuronic acid (GlcA) side chains. All the GlcA side chains of xylan in wood of Populus trichocarpa are methylated, which is different from Arabidopsis xylan in which about 60% of GlcA side chains are methylated. Genes responsible for methylation of GlcA side chains in Populus xylan have not been identified. Here, we report genetic and biochemical analyses of four DUF579 domain-containing proteins, PtrGXM1, PtrGXM2, PtrGXM3 and PtrGXM4, from Populus trichocarpa and their roles in GlcA methylation in xylan. The PtrGXM genes were found to be highly expressed in wood-forming cells and their encoded proteins were shown to be localized in the Golgi. When overexpressed in the Arabidopsis gxm1/2/3 triple mutant, PtrGXMs were able to partially complement the mutant phenotypes including defects in glucuronoxylan methyltransferase activity and GlcA methylation in xylan, indicating that PtrGXMs most likely function as glucuronoxylan methyltransferases. Direct evidence was provided by enzymatic analysis of recombinant PtrGXM proteins showing that they possessed a methyltransferase activity capable of transferring the methyl group onto GlcA-substituted xylooligomers. Kinetic analysis showed that PtrGXMs exhibited differential affinities toward the GlcA-substituted xylooligomer acceptor with PtrGXM3 and PtrGXM4 having 10 times higher K m values than PtrGXM1 and PtrGXM2. Together, these findings indicate that PtrGXMs are methyltransferases mediating GlcA methylation in Populus xylan during wood formation.

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Kinetic properties of the PtrGXM methyltransferase activities.Recombinant PtrGXM proteins were assayed for the methyltransferase activity in the presence of various concentrations of the (GlcA)Xyl4 acceptor. The results were analyzed by Lineweaver-Burk plots to determine the Km and Vmax values.
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pone-0087370-g011: Kinetic properties of the PtrGXM methyltransferase activities.Recombinant PtrGXM proteins were assayed for the methyltransferase activity in the presence of various concentrations of the (GlcA)Xyl4 acceptor. The results were analyzed by Lineweaver-Burk plots to determine the Km and Vmax values.

Mentions: We further analyzed the biochemical properties of the recombinant PtrGXMs. The glucuronoxylan methyltransferase activities of PtrGXMs were both time- and protein concentration-dependent (Fig. 10A, B). The optimal temperature for the reactions was 37°C for PtrGXM1 and PtrGXM2 and 45°C for PtrGXM3 and PtrGXM4 (Fig. 10C). To examine the substrate binding affinities of PtrGXMs, we investigated their kinetic properties using different concentrations of the GlcA-substituted Xyl4 acceptor. The Km and Vmax values were calculated by Lineweaver-Burk analysis. It was found that the transfer of the methyl group onto GlcA-substituted Xyl4 by PtrGXMs was dependent on the acceptor concentration (Fig. 11) with apparent Km values of 3.25, 3.98, 0.30 and 0.29 mM and Vmax of 145, 123, 353 and 737 pmol/min/mg protein for PtrGXM1, PtrGXM2, PtrGXM3, and PtrGXM4, respectively. These results indicate that PtrGXM3 and PtrGXM4 exhibit much higher substrate affinities for the GlcA-substituted Xyl4 acceptor than PtrGXM1 and PtrGXM2.


Identification and biochemical characterization of four wood-associated glucuronoxylan methyltransferases in Populus.

Yuan Y, Teng Q, Zhong R, Ye ZH - PLoS ONE (2014)

Kinetic properties of the PtrGXM methyltransferase activities.Recombinant PtrGXM proteins were assayed for the methyltransferase activity in the presence of various concentrations of the (GlcA)Xyl4 acceptor. The results were analyzed by Lineweaver-Burk plots to determine the Km and Vmax values.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921138&req=5

pone-0087370-g011: Kinetic properties of the PtrGXM methyltransferase activities.Recombinant PtrGXM proteins were assayed for the methyltransferase activity in the presence of various concentrations of the (GlcA)Xyl4 acceptor. The results were analyzed by Lineweaver-Burk plots to determine the Km and Vmax values.
Mentions: We further analyzed the biochemical properties of the recombinant PtrGXMs. The glucuronoxylan methyltransferase activities of PtrGXMs were both time- and protein concentration-dependent (Fig. 10A, B). The optimal temperature for the reactions was 37°C for PtrGXM1 and PtrGXM2 and 45°C for PtrGXM3 and PtrGXM4 (Fig. 10C). To examine the substrate binding affinities of PtrGXMs, we investigated their kinetic properties using different concentrations of the GlcA-substituted Xyl4 acceptor. The Km and Vmax values were calculated by Lineweaver-Burk analysis. It was found that the transfer of the methyl group onto GlcA-substituted Xyl4 by PtrGXMs was dependent on the acceptor concentration (Fig. 11) with apparent Km values of 3.25, 3.98, 0.30 and 0.29 mM and Vmax of 145, 123, 353 and 737 pmol/min/mg protein for PtrGXM1, PtrGXM2, PtrGXM3, and PtrGXM4, respectively. These results indicate that PtrGXM3 and PtrGXM4 exhibit much higher substrate affinities for the GlcA-substituted Xyl4 acceptor than PtrGXM1 and PtrGXM2.

Bottom Line: The PtrGXM genes were found to be highly expressed in wood-forming cells and their encoded proteins were shown to be localized in the Golgi.Kinetic analysis showed that PtrGXMs exhibited differential affinities toward the GlcA-substituted xylooligomer acceptor with PtrGXM3 and PtrGXM4 having 10 times higher K m values than PtrGXM1 and PtrGXM2.Together, these findings indicate that PtrGXMs are methyltransferases mediating GlcA methylation in Populus xylan during wood formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
Wood is one of the promising bioenergy feedstocks for lignocellulosic biofuel production. Understanding how wood components are synthesized will help us design strategies for better utilization of wood for biofuel production. One of the major wood components is xylan, in which about 10% of xylosyl residues are substituted with glucuronic acid (GlcA) side chains. All the GlcA side chains of xylan in wood of Populus trichocarpa are methylated, which is different from Arabidopsis xylan in which about 60% of GlcA side chains are methylated. Genes responsible for methylation of GlcA side chains in Populus xylan have not been identified. Here, we report genetic and biochemical analyses of four DUF579 domain-containing proteins, PtrGXM1, PtrGXM2, PtrGXM3 and PtrGXM4, from Populus trichocarpa and their roles in GlcA methylation in xylan. The PtrGXM genes were found to be highly expressed in wood-forming cells and their encoded proteins were shown to be localized in the Golgi. When overexpressed in the Arabidopsis gxm1/2/3 triple mutant, PtrGXMs were able to partially complement the mutant phenotypes including defects in glucuronoxylan methyltransferase activity and GlcA methylation in xylan, indicating that PtrGXMs most likely function as glucuronoxylan methyltransferases. Direct evidence was provided by enzymatic analysis of recombinant PtrGXM proteins showing that they possessed a methyltransferase activity capable of transferring the methyl group onto GlcA-substituted xylooligomers. Kinetic analysis showed that PtrGXMs exhibited differential affinities toward the GlcA-substituted xylooligomer acceptor with PtrGXM3 and PtrGXM4 having 10 times higher K m values than PtrGXM1 and PtrGXM2. Together, these findings indicate that PtrGXMs are methyltransferases mediating GlcA methylation in Populus xylan during wood formation.

Show MeSH
Related in: MedlinePlus