Abstract
The chemical complexity and biological activity of the glycopeptide antibiotics (GPAs) stems from their unique crosslinked structure, which is generated by the actions of cytochrome P450 (Oxy) enzymes that affect the crosslinking of aromatic side chains of amino acid residues contained within the GPA heptapeptide precursor. Given the crucial role peptide cyclisation plays in GPA activity, the characterisation of this process is of great importance in understanding the biosynthesis of these important antibiotics. Here, we report the cyclisation activity and crystal structure of StaF, the D-O-E ring forming Oxy enzyme from A47934 biosynthesis. Our results show that the specificity of StaF is reduced when compared to Oxy enzymes catalysing C-O-D ring formation and that this activity relies on interactions with the non-ribosomal peptide synthetase via the X-domain. Despite the interaction of StaF with the A47934 X-domain being weaker than for the preceding Oxy enzyme StaH, StaF retains higher levels of in vitro activity: we postulate that this is due to the ability of the StaF/X-domain complex to allow substrate reorganisation after initial complex formation has occurred. These results highlight the importance of testing different peptide/protein carrier constructs for in vitro GPA cyclisation assays and show that different Oxy homologues can display significantly different catalytic propensities despite their overall similarities.
| Original language | English |
|---|---|
| Pages (from-to) | 2849-2864 |
| Number of pages | 16 |
| Journal | Beilstein Journal of Organic Chemistry |
| Volume | 12 |
| DOIs | |
| Publication status | Published - 27 Dec 2016 |
Keywords
- Crystal structure
- Cytochrome P450
- Glycopeptide antibiotic
- Peptide
- Phenolic coupling
Cite this
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Biochemical and structural characterisation of the second oxidative crosslinking step during the biosynthesis of the glycopeptide antibiotic A47934. / Ulrich, Veronika; Brieke, Clara; Cryle, Max J.
In: Beilstein Journal of Organic Chemistry, Vol. 12, 27.12.2016, p. 2849-2864.Research output: Contribution to journal › Article › Research › peer-review
TY - JOUR
T1 - Biochemical and structural characterisation of the second oxidative crosslinking step during the biosynthesis of the glycopeptide antibiotic A47934
AU - Ulrich, Veronika
AU - Brieke, Clara
AU - Cryle, Max J.
PY - 2016/12/27
Y1 - 2016/12/27
N2 - The chemical complexity and biological activity of the glycopeptide antibiotics (GPAs) stems from their unique crosslinked structure, which is generated by the actions of cytochrome P450 (Oxy) enzymes that affect the crosslinking of aromatic side chains of amino acid residues contained within the GPA heptapeptide precursor. Given the crucial role peptide cyclisation plays in GPA activity, the characterisation of this process is of great importance in understanding the biosynthesis of these important antibiotics. Here, we report the cyclisation activity and crystal structure of StaF, the D-O-E ring forming Oxy enzyme from A47934 biosynthesis. Our results show that the specificity of StaF is reduced when compared to Oxy enzymes catalysing C-O-D ring formation and that this activity relies on interactions with the non-ribosomal peptide synthetase via the X-domain. Despite the interaction of StaF with the A47934 X-domain being weaker than for the preceding Oxy enzyme StaH, StaF retains higher levels of in vitro activity: we postulate that this is due to the ability of the StaF/X-domain complex to allow substrate reorganisation after initial complex formation has occurred. These results highlight the importance of testing different peptide/protein carrier constructs for in vitro GPA cyclisation assays and show that different Oxy homologues can display significantly different catalytic propensities despite their overall similarities.
AB - The chemical complexity and biological activity of the glycopeptide antibiotics (GPAs) stems from their unique crosslinked structure, which is generated by the actions of cytochrome P450 (Oxy) enzymes that affect the crosslinking of aromatic side chains of amino acid residues contained within the GPA heptapeptide precursor. Given the crucial role peptide cyclisation plays in GPA activity, the characterisation of this process is of great importance in understanding the biosynthesis of these important antibiotics. Here, we report the cyclisation activity and crystal structure of StaF, the D-O-E ring forming Oxy enzyme from A47934 biosynthesis. Our results show that the specificity of StaF is reduced when compared to Oxy enzymes catalysing C-O-D ring formation and that this activity relies on interactions with the non-ribosomal peptide synthetase via the X-domain. Despite the interaction of StaF with the A47934 X-domain being weaker than for the preceding Oxy enzyme StaH, StaF retains higher levels of in vitro activity: we postulate that this is due to the ability of the StaF/X-domain complex to allow substrate reorganisation after initial complex formation has occurred. These results highlight the importance of testing different peptide/protein carrier constructs for in vitro GPA cyclisation assays and show that different Oxy homologues can display significantly different catalytic propensities despite their overall similarities.
KW - Crystal structure
KW - Cytochrome P450
KW - Glycopeptide antibiotic
KW - Peptide
KW - Phenolic coupling
UR - http://www.scopus.com/inward/record.url?scp=85008196922&partnerID=8YFLogxK
U2 - 10.3762/bjoc.12.284
DO - 10.3762/bjoc.12.284
M3 - Article
AN - SCOPUS:85008196922
VL - 12
SP - 2849
EP - 2864
JO - Beilstein Journal of Organic Chemistry
JF - Beilstein Journal of Organic Chemistry
SN - 1860-5397
ER -