Engineering a Monolignol 4-O-Methyltransferase with High Selectivity for the Condensed Lignin Precursor Coniferyl Alcohol

Cai, Yuanheng; Bhuiya, Mohammad-Wadud; Shanklin, John; Liu, Changjun

doi:10.1074/jbc.m115.684217

Cited by 13 publications

(9 citation statements)

References 38 publications

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“…Directed evolution is also a powerful tool for engineering protein domain specificity over cognate target residues or for developing enzymes with different activities (Figure b). An example comes from Cai et al, who in 2015 showed how directed evolution of the plant enzyme monolignol 4- O -methyltransferase could be used to change its substrate specificity with consequent ability to improve lignin digestion and conversion . More recently, Dai et al.…”

Section: Applications In Biological Engineeringmentioning

confidence: 99%

“…An example comes from Cai et al, who in 2015 showed how directed evolution of the plant enzyme monolignol 4-Omethyltransferase could be used to change its substrate specificity with consequent ability to improve lignin digestion and conversion. 121 More recently, Dai et al engineered the histidine methyltransferase SETD3, changing its target specificity from histidine to lysine. 122 By comparing the active site of SETD3 with the ones of the lysine methyltransferases SETD6 and LSMT, the authors individuated two amino acids, Asn55 and Trp273, responsible for histidine specificity.…”

Section: ■ Applications In Biological Engineeringmentioning

confidence: 99%

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Non-Histone Protein Methylation: Biological Significance and Bioengineering Potential

et al. 2021

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Protein methylation is a key post-translational modification whose effects on gene expression have been intensively studied over the last two decades. Recently, renewed interest in non-histone protein methylation has gained momentum for its role in regulating important cellular processes and the activity of many proteins, including transcription factors, enzymes, and structural complexes. The extensive and dynamic role that protein methylation plays within the cell also highlights its potential for bioengineering applications. Indeed, while synthetic histone protein methylation has been extensively used to engineer gene expression, engineering of non-histone protein methylation has not been fully explored yet. Here, we report the latest findings, highlighting how non-histone protein methylation is fundamental for certain cellular functions and is implicated in disease, and review recent efforts in the engineering of protein methylation.

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Section: Applications In Biological Engineeringmentioning

confidence: 99%

Section: ■ Applications In Biological Engineeringmentioning

confidence: 99%

Non-Histone Protein Methylation: Biological Significance and Bioengineering Potential

et al. 2021

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“…The change of a few residues at the OMT-I substrate-binding site could modify their regioselectivity and substrate specificity. Other rational and semi-rational designs have been performed with CbIEMT1, increasing its substrate scope [ 37 ] or generating a highly specific enzyme [ 38 ].…”

Section: Resultsmentioning

confidence: 99%

Rational Design of Resveratrol O-methyltransferase for the Production of Pinostilbene

Herrera

Chánique

Martínez-Márquez

et al. 2021

IJMS

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Pinostilbene is a monomethyl ether analog of the well-known nutraceutical resveratrol. Both compounds have health-promoting properties, but the latter undergoes rapid metabolization and has low bioavailability. O-methylation improves the stability and bioavailability of resveratrol. In plants, these reactions are performed by O-methyltransferases (OMTs). Few efficient OMTs that monomethylate resveratrol to yield pinostilbene have been described so far. Here, we report the engineering of a resveratrol OMT from Vitis vinifera (VvROMT), which has the highest catalytic efficiency in di-methylating resveratrol to yield pterostilbene. In the absence of a crystal structure, we constructed a three-dimensional protein model of VvROMT and identified four critical binding site residues by applying different in silico approaches. We performed point mutations in these positions generating W20A, F24A, F311A, and F318A variants, which greatly reduced resveratrol’s enzymatic conversion. Then, we rationally designed eight variants through comparison of the binding site residues with other stilbene OMTs. We successfully modified the native substrate selectivity of VvROMT. Variant L117F/F311W showed the highest conversion to pinostilbene, and variant L117F presented an overall increase in enzymatic activity. Our results suggest that VvROMT has potential for the tailor-made production of stilbenes.

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“…Furthermore, two tested MOMT variants exhibit different effects on lignin and phenolic biosynthesis in planta. MOMT4, which favours methylating the S-type sinapyl alcohol over the G-type coniferyl alcohol in vitro (Bhuiya and Liu, 2010;Zhang et al, 2012), suppresses more S lignin deposition in transgenic rice, resulting in large reductions in the S/G lignin ratios as determined by both thioacidolysis (Figure 5c) and 2D NMR (Figure 6c), whereas expressing MOMT9, a variant that more prefers modifying G monomers over S monomers in vitro due to its smaller substrate binding pocket (Cai et al, 2015), leads to a less prominent effect on the S/G lignin ratio reductions (Figures 5c and 6c), and concomitantly the sole production of the soluble 4-O-methylated FA-derived metabolites (Figure 2). These results demonstrate that both MOMT variants function properly in grass species and are in line with their in vitro structural and catalytic properties.…”

Section: Discussionmentioning

confidence: 99%

Simultaneous suppression of lignin, tricin and wall‐bound phenolic biosynthesis via the expression of monolignol 4‐O‐methyltransferases in rice

Dwivedi,

Yamamoto,

Zhao

et al. 2023

Plant Biotechnology Journal

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SummaryGrass lignocelluloses feature complex compositions and structures. In addition to the presence of conventional lignin units from monolignols, acylated monolignols and flavonoid tricin also incorporate into lignin polymer; moreover, hydroxycinnamates, particularly ferulate, cross‐link arabinoxylan chains with each other and/or with lignin polymers. These structural complexities make grass lignocellulosics difficult to optimize for effective agro‐industrial applications. In the present study, we assess the applications of two engineered monolignol 4‐O‐methyltransferases (MOMTs) in modifying rice lignocellulosic properties. Two MOMTs confer regiospecific para‐methylation of monolignols but with different catalytic preferences. The expression of MOMTs in rice resulted in differential but drastic suppression of lignin deposition, showing more than 50% decrease in guaiacyl lignin and up to an 90% reduction in syringyl lignin in transgenic lines. Moreover, the levels of arabinoxylan‐bound ferulate were reduced by up to 50%, and the levels of tricin in lignin fraction were also substantially reduced. Concomitantly, up to 11 μmol/g of the methanol‐extractable 4‐O‐methylated ferulic acid and 5–7 μmol/g 4‐O‐methylated sinapic acid were accumulated in MOMT transgenic lines. Both MOMTs in vitro displayed discernible substrate promiscuity towards a range of phenolics in addition to the dominant substrate monolignols, which partially explains their broad effects on grass phenolic biosynthesis. The cell wall structural and compositional changes resulted in up to 30% increase in saccharification yield of the de‐starched rice straw biomass after diluted acid‐pretreatment. These results demonstrate an effective strategy to tailor complex grass cell walls to generate improved cellulosic feedstocks for the fermentable sugar‐based production of biofuel and bio‐chemicals.

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Engineering a Monolignol 4-O-Methyltransferase with High Selectivity for the Condensed Lignin Precursor Coniferyl Alcohol

Cited by 13 publications

References 38 publications

Non-Histone Protein Methylation: Biological Significance and Bioengineering Potential

Non-Histone Protein Methylation: Biological Significance and Bioengineering Potential

Rational Design of Resveratrol O-methyltransferase for the Production of Pinostilbene

Simultaneous suppression of lignin, tricin and wall‐bound phenolic biosynthesis via the expression of monolignol 4‐O‐methyltransferases in rice

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