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. 2009 Nov 3;48(43):10467-72.
doi: 10.1021/bi9013165.

The ATP-dependent amide ligases DdaG and DdaF assemble the fumaramoyl-dipeptide scaffold of the dapdiamide antibiotics

Marie A Hollenhorst  1 , Jon Clardy, Christopher T Walsh
Affiliations

The ATP-dependent amide ligases DdaG and DdaF assemble the fumaramoyl-dipeptide scaffold of the dapdiamide antibiotics

Marie A Hollenhorst et al. Biochemistry. .

Erratum in

  • Biochemistry. 2010 Jul 27;49(29):6296

Abstract

The enzymes DdaG and DdaF, encoded in the Pantoea agglomerans dapdiamide antibiotic biosynthetic gene cluster, when expressed in Escherichia coli, form the tandem amide bonds of the dapdiamide scaffold at the expense of ATP cleavage. DdaG uses fumarate, 2,3-diaminopropionate (DAP), and ATP to make fumaroyl-AMP transiently on the way to the N(beta)-fumaroyl-DAP regioisomer. Then DdaF acts as a second ATP-dependent amide ligase, but this enzyme cleaves ATP to ADP and P(i) during amide bond formation. However, DdaF will not accept N(beta)-fumaroyl-DAP; the enzyme requires the fumaroyl moiety to be first converted to the fumaramoyl half-amide in N(beta)-fumaramoyl-DAP. DdaF adds Val, Ile, or Leu to the carboxylate of fumaramoyl-DAP to make dapdiamide A, B, or C, respectively. Thus, to build the dapdiamide antibiotic scaffold, amidation must occur on the fumaroyl-DAP scaffold, after DdaG action but before DdaF catalysis. This is an unusual instance of two ligases acting sequentially in untemplated amide bond formations using attack of substrate carboxylates at P(alpha) (AMP-forming) and then at P(gamma) (ADP-forming) of ATP cosubstrates.

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Figures

Figure 1
Figure 1
A) The dapdiamide family of antibiotics and Nβ-fumaramoyl-DAP. B) Dapdiamide-related natural products. C) Proposed mechanism of GlcN-6-P synthase inhibition by Nβ-fumaramoyl-DAP.
Figure 2
Figure 2
Characterization of DdaG activity. A) HPLC analysis showing ATP-dependent ligation of fumarate and DAP by DdaG. B) ATP/[32P]-PPi exchange data. Graph shows counts per minute arising from [32P]-ATP that is adsorbed to charcoal after incubation of DdaG, [32P]-PPi, unlabeled ATP, and carboxylate substrate. *Activity with fumaramate results from 2–5% contamination with fumarate.2 C) Coelution of the FMOC-derivatized DdaG product with FMOC-Nβ-fumaroyl-DAP.
Figure 3
Figure 3
Relative rates of ADP formation on incubation of DdaF with potential carboxylate substrates and 2 mM Val.
Figure 4
Figure 4
HPLC analysis showing ATP-dependent ligation of Nβ-fumaramoyl-DAP and Val by DdaF to form dapdiamide A.
Scheme 1
Scheme 1
Biosynthetic route to dapdiamides A-C. The grayed-out step has not been biochemically validated.
See this image and copyright information in PMC

References

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