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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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