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Table 2 Significance of the putative residues of TaOMT2 involved in binding ans/or catalysis and changes in the properties of their mutant proteins1

From: Structure-function relationships of wheat flavone O-methyltransferase: Homology modeling and site-directed mutagenesis

Wild type Significance Mutant Enzyme activity2 Properties of mutant proteins
residues   proteins (%) Km Vmax Kcat/Km  
Control    100 59.5 110 74  
D263 Important residue for substrate binding; forms H-bonds with All OH groups of tricetin D263E 4.01     Severe loss of activity is due to a conflict between the catalytic His262-imidazole group and Glu-CH2
   D263I 0.08     Ile263 can not form a H-bond with 3'-OH group
   D263N 84.33 128.2 20.3 63 Slight decrease in activity due to a decreased electronegativity of Asn-N compared to Asp-O, that affects charge transfer to tricetin-OH groups
E290 H-bonds with tricetin 4'-OH; forms an H-bonding network with neighboring residues, esp. E290-COO- and H262-backbone-NH E290I 1.7     Loss of activity is due to the fact that Ile can not form a H-bond with the 4'-OH of tricetin
   E290Q 0.06     This mutation results in a more extensive H-bonding that hinders charge transfer and affects B-ring flexibility
W259 H-bonds with selgin 4'-OH and forms a H-bonding network with neighboring residues W259A 79.23 131.0 17.02 5.20 Ala can maintain the H-bonding network between Trp259, Glu290 and His262, wheras Tyr cannot
   W259Y 49.13 170.1 9.90 2.33  
E322 H-bonds with tricetin 5'-OH. E322I 54.33 193.17 30.56 6.33 Loss of charge or a change in the side chain affects H-bonding with the neighboring residues, especially His262
   E322Q 40.3     
G305 H-bonds with selgin 7-OH; important residue for substrate positioning G305S 63.33 118.41 21.65 7.31 Change in polarity is less effective than chain length on catalytic activity.
   G305A 0.14     Loss of activity due to loss of H-bonding with the amide group of the neighboring Asn348
N124 H-bonds with O-4/O-5 of all substrates in order to orient them to the most favorable position N124I 1.8     Resuled in a decreased substrate binding but not protein folding. Both mutations disrupt H-bonding with 5-OH group of tricetin
   N124Q 4.1     
H262 Putative catalytic base involved in deprotonation of tricetin hydroxyl groups H262R 0.01     Resulted in almost complete loss of protein expression; all mutant proteins lack imidazole ring that is critical for proton flow among His262, Asp263 and the substrate
   H262L 0.96     
   H262F 1.06     
  1. 1Site directed mutagenesis and preparation of mutant proteins were conducted as described in the Methods section.
  2. 2Enzyme activity with tricetin as substrate is expressed in terms of % relative activity as compared with wild-type protein, Km values (μM), Vmax (pkat.mg-1; pkat, the catalytic activity that raises the reaction rate by one pmol.s-1), and catalytic efficiency (Kcat/Km nM-1.s-1), respectively.
  3. 3HPLC analysis of the enzyme reaction products showed tricin as the predominant product, with a trace amount of trimethyltricetin (see Additional Fig. 4B).
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BMC Plant Biology

ISSN: 1471-2229

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