Information on EC 6.3.2.47 - dapdiamide synthase

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Biosynthesis of secondary metabolites, Biosynthesis of various secondary metabolites - part 3

{[(2E)-4-amino-4-oxobut-2-enoyl]amino}-L-alanine:L-valine ligase (ADP-forming)

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ATP + 3-({[(2R,3R)-3-carbamoyloxiran-2-yl]carbonyl}amino)-L-alanine + L-valine = ADP + phosphate + 3-({[(2R,3R)-3-carbamoyloxiran-2-yl]carbonyl}amino)-L-alanyl-L-valine

(4)

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ATP + 3-{[(2E)-4-amino-4-oxobut-2-enoyl]amino}-L-alanine + L-isoleucine = ADP + phosphate + 3-{[(2E)-4-amino-4-oxobut-2-enoyl]amino}-L-alanyl-L-isoleucine

(2)

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ATP + 3-{[(2E)-4-amino-4-oxobut-2-enoyl]amino}-L-alanine + L-leucine = ADP + phosphate + 3-{[(2E)-4-amino-4-oxobut-2-enoyl]amino}-L-alanyl-L-leucine

(3)

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ATP + 3-{[(2E)-4-amino-4-oxobut-2-enoyl]amino}-L-alanine + L-valine = ADP + phosphate + 3-{[(2E)-4-amino-4-oxobut-2-enoyl]amino}-L-alanyl-L-valine

(1)

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BRENDA

dapdiamides biosynthesis

KEGG

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Biosynthesis of various secondary metabolites - part 3

3-{[(2E)-4-amino-4-oxobut-2-enoyl]amino}-L-alanine:L-valine ligase (ADP-forming)

The enzyme, characterized from the bacterium Pantoea agglomerans, is involved in biosynthesis of dapdiamide tripeptide antibiotics, a family of fumaramoyl- and epoxysuccinamoyl-peptides named for the presence of an (S)-2,3-diaminopropanoate (DAP) moiety and two amide linkages in their scaffold.

ATP + 3-((((2R,3R)-3-carboxyoxiran-2-yl)carbonyl)amino)-L-alanine + L-valine

ADP + phosphate + 3-((((2R,3R)-3-carboxyoxiran-2-yl)carbonyl)amino)-L-alanyl-L-valine

i.e. N-beta-epoxysuccinamoyl-2,3-diaminopropionate. DdaF can catalyze the ligation of both Nbeta-trans-epoxysuccinamoyl-diaminopropionate diastereomers to Val to produce the Nbeta-trans-epoxysuccinamoyl-diaminopropionate-L-valine dipeptide antibiotics. DdaF uses (R,R)-Nbeta-trans-epoxysuccinamoyl-diaminopropionate as a saturable substrate, whereas saturation is not achieved with (S,S)-Nbeta-trans-epoxysuccinamoyl-diaminopropionate at concentrations up to 590 microM

product is a natural antibiotic

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ATP + 3-[[(2E)-4-amino-4-oxobut-2-enoyl]amino]-L-alanine + L-isoleucine

ADP + phosphate + 3-(((2E)-4-amino-4-oxobut-2-enoyl)amino)-L-alanyl-L-isoleucine

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ATP + 3-[[(2E)-4-amino-4-oxobut-2-enoyl]amino]-L-alanine + L-leucine

ADP + phosphate + 3-(((2E)-4-amino-4-oxobut-2-enoyl)amino)-L-alanyl-L-leucine

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ATP + 3-[[(2E)-4-amino-4-oxobut-2-enoyl]amino]-L-alanine + L-valine

ADP + phosphate + 3-(((2E)-4-amino-4-oxobut-2-enoyl)amino)-L-alanyl-L-valine

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ATP + 3-[[(2E)-4-amino-4-oxobut-2-enoyl]amino]-L-alanine + L-valine

ADP + phosphate + 3-[[(2E)-4-amino-4-oxobut-2-enoyl]amino]-L-alanyl-L-valine

i.e. Nbeta-fumaramoyl-diaminopropionate

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additional information

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DdaF will not accept Nbeta-fumaroyl-diaminopropionate or Nalpha-fumaramoyl-diaminopropionate. The enzyme requires the fumaroyl moiety to be first converted to the fumaramoyl half amide in Nbeta-fumaramoyl-diaminopropionate

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Nbeta-fumaramoyl-diaminopropionate

substrate inhibition above 0.5 mM

0.053

3-((((2R,3R)-3-carboxyoxiran-2-yl)carbonyl)amino)-L-alanine

pH 8.0, 22°C

0.072

3-[[(2E)-4-amino-4-oxobut-2-enoyl]amino]-L-alanine

pH 8.0, 22°C

0.83

L-isoleucine

pH 8.0, 22°C

7.82

L-leucine

pH 8.0, 22°C

0.26

L-valine

pH 8.0, 22°C

3

3-((((2R,3R)-3-carboxyoxiran-2-yl)carbonyl)amino)-L-alanine

pH 8.0, 22°C

0.3

3-[[(2E)-4-amino-4-oxobut-2-enoyl]amino]-L-alanine

pH 8.0, 22°C

56.7

3-((((2R,3R)-3-carboxyoxiran-2-yl)carbonyl)amino)-L-alanine

pH 8.0, 22°C

4.83

3-[[(2E)-4-amino-4-oxobut-2-enoyl]amino]-L-alanine

pH 8.0, 22°C

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UniProt

physiological function

gene DdaF belongs to the dapdiamide antibiotic biosynthetic gene cluster. DdaF acts as a second ATP-dependent amide ligase, cleaving ATP to ADP and Pi during amide bond formation. DdaF adds Val, Ile, or Leu to the carboxylate of fumaramoyl-2,3-diaminopropionate to make the three dapdiamides A-C. To build the dapdiamide antibiotic scaffold, amidation must occur on the fumaroyl-diaminopropionate scaffold, after DdaG action but before DdaF catalysis

expression in Escherichia coli