PDBsum entry 3zk5

Oxidoreductase

PDB id: 3zk5

Contents

Protein chains

397 a.a.

Ligands

HEM ×2

Z18 ×2

Waters ×524

PDB id:

3zk5

Name:

Oxidoreductase

Title:

Pikc d50n mutant bound to the 10-dml analog with the 3-(n,n- dimethylamino)ethanoate anchoring group

Structure:

Cytochrome p450 hydroxylase pikc. Chain: a, b. Synonym: pikc. Engineered: yes. Mutation: yes. Other_details: thiolate link between c 354 and hem 1407

Source:

Streptomyces venezuelae. Organism_taxid: 54571. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.89Å R-factor: 0.200 R-free: 0.267

Authors:

L.M.Podust

Key ref:

S.Negretti et al. (2014). Directing group-controlled regioselectivity in an enzymatic C-H bond oxygenation. J Am Chem Soc, 136, 4901-4904. PubMed id: 24627965 DOI: 10.1021/ja5016052

Date:

21-Jan-13 Release date: 29-Jan-14

Protein chains

O87605  (PIKC_STRVZ) -  Cytochrome P450 monooxygenase PikC from Streptomyces venezuelae

Seq: 416 a.a.

Struc: 397 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.1.14.15.33 - pikromycin synthase.
• Reaction:
  1. narbomycin + 2 reduced [2Fe-2S]-[ferredoxin] + O2 + 2 H⁺ = pikromycin + 2 oxidized [2Fe-2S]-[ferredoxin] + H2O
  2. narbomycin + 2 reduced [2Fe-2S]-[ferredoxin] + O2 + 2 H⁺ = neopikromycin + 2 oxidized [2Fe-2S]-[ferredoxin] + H2O
  3. narbomycin + 4 reduced [2Fe-2S]-[ferredoxin] + 2 O2 + 4 H⁺ = novapikromycin + 4 oxidized [2Fe-2S]-[ferredoxin] + 2 H2O
  4. 10-deoxymethymycin + 2 reduced [2Fe-2S]-[ferredoxin] + O2 + 2 H⁺ = methymycin + 2 oxidized [2Fe-2S]-[ferredoxin] + H2O
  5. 10-deoxymethymycin + 2 reduced [2Fe-2S]-[ferredoxin] + O2 + 2 H⁺ = neomethymycin + 2 oxidized [2Fe-2S]-[ferredoxin] + H2O
  6. 10-deoxymethymycin +

narbomycin + 2 × reduced [2Fe-2S]-[ferredoxin] (Bound ligand (Het Group name = Z18) matches with 61.54% similarity) + O2 + 2 × H(+) = pikromycin + 2 × oxidized [2Fe-2S]-[ferredoxin] + H2O

narbomycin + 2 × reduced [2Fe-2S]-[ferredoxin] (Bound ligand (Het Group name = Z18) matches with 61.54% similarity) + O2 + 2 × H(+) = neopikromycin + 2 × oxidized [2Fe-2S]-[ferredoxin] + H2O

narbomycin + 4 × reduced [2Fe-2S]-[ferredoxin] (Bound ligand (Het Group name = Z18) matches with 61.54% similarity) + 2 × O2 + 4 × H(+) = novapikromycin + 4 × oxidized [2Fe-2S]-[ferredoxin] + 2 × H2O

10-deoxymethymycin (Bound ligand (Het Group name = Z18) matches with 68.57% similarity) + 2 × reduced [2Fe-2S]-[ferredoxin] + O2 + 2 × H(+) = methymycin + 2 × oxidized [2Fe-2S]-[ferredoxin] + H2O

10-deoxymethymycin (Bound ligand (Het Group name = Z18) matches with 68.57% similarity) + 2 × reduced [2Fe-2S]-[ferredoxin] + O2 + 2 × H(+) = neomethymycin + 2 × oxidized [2Fe-2S]-[ferredoxin] + H2O

10-deoxymethymycin (Bound ligand (Het Group name = Z18) matches with 68.57% similarity) +

• Cofactor: Heme-thiolate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1021/ja5016052

J Am Chem Soc 136:4901-4904 (2014)

PubMed id: 24627965

Directing group-controlled regioselectivity in an enzymatic C-H bond oxygenation.

S.Negretti, A.R.Narayan, K.C.Chiou, P.M.Kells, J.L.Stachowski, D.A.Hansen, L.M.Podust, J.Montgomery, D.H.Sherman.

ABSTRACT

Highly regioselective remote hydroxylation of a natural product scaffold is demonstrated by exploiting the anchoring mechanism of the biosynthetic P450 monooxygenase PikCD50N-RhFRED. Previous studies have revealed structural and biochemical evidence for the role of a salt bridge between the desosamine N,N-dimethylamino functionality of the natural substrate YC-17 and carboxylate residues within the active site of the enzyme, and selectivity in subsequent C-H bond functionalization. In the present study, a substrate-engineering approach was conducted that involves replacing desosamine with varied synthetic N,N-dimethylamino anchoring groups. We then determined their ability to mediate enzymatic total turnover numbers approaching or exceeding that of the natural sugar, while enabling ready introduction and removal of these amino anchoring groups from the substrate. The data establish that the size, stereochemistry, and rigidity of the anchoring group influence the regioselectivity of enzymatic hydroxylation. The natural anchoring group desosamine affords a 1:1 mixture of regioisomers, while synthetic anchors shift YC-17 analogue C-10/C-12 hydroxylation from 20:1 to 1:4. The work demonstrates the utility of substrate engineering as an orthogonal approach to protein engineering for modulation of regioselective C-H functionalization in biocatalysis.