Mutational analysis of 4-coumarate:CoA ligase identifies functionally important amino acids and verifies its close relationship to other adenylate-forming enzymes.
4-Coumarate:coenzyme A ligase (4CL) is a key enzyme of general phenylpropanoid metabolism which provides the precursors for a large variety of important plant secondary products, such as lignin, flavonoids, or phytoalexins. To identify amino acids important for 4CL activity, eight mutations were introduced into Arabidopsis thaliana At4CL2. Determination of specific activities and K(m) values for ATP and caffeate of the heterologously expressed and purified proteins identified four distinct classes of mutants: enzymes with little or no catalytic activity; enzymes with greatly reduced activity but wild-type K(m) values; enzymes with drastically altered K(m) values; and enzymes with almost wild-type properties. The latter class includes replacement of a cysteine residue which is strictly conserved in 4CLs and had previously been assumed to be directly involved in catalysis. These results substantiate the close relationship between 4CL and other adenylate-forming enzymes such as luciferases, peptide synthetases, and fatty acyl-CoA synthetases.
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Max-Planck-Institut für Züchtungsforschung, Abteilung Biochemie, Carl-von-Linné-Weg 10, 50829, Köln, Germany.
Büttner
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Ehlting
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Hahlbrock
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Kombrink
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D000249:Adenosine Monophosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology*; D001665:Binding Sites; D002109:Caffeic Acids / Q000378:metabolism; D002384:Catalysis; D003066:Coenzyme A Ligases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017124:Conserved Sequence / Q000235:genetics; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D009711:Nucleotides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013816:Thermodynamics
D000596:Amino Acids; D002109:Caffeic Acids; D009711:Nucleotides; D011993:Recombinant Fusion Proteins; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; C019943:4-coumarate-CoA ligase; D003545:Cysteine; C040048:caffeic acid