Information on EC 1.14.15.33 - pikromycin synthase

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CYP107L2

EC 1.14.13.185

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formerly

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erythromycin C-12 hydroxylase

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PikC

3 entries

10-deoxymethymycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2 = methymycin + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

10-deoxymethymycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2 = neomethymycin + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

10-deoxymethymycin + 4 reduced ferredoxin [iron-sulfur] cluster + 4 H+ + 2 O2 = novamethymycin + 4 oxidized ferredoxin [iron-sulfur] cluster + 2 H2O

narbomycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2 = neopikromycin + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

narbomycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2 = pikromycin + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

narbomycin + 4 reduced ferredoxin [iron-sulfur] cluster + 4 H+ + 2 O2 = novapikromyin + 4 oxidized ferredoxin [iron-sulfur] cluster + 2 H2O

macrolide antibiotic biosynthesis

BRENDA

KEGG

Biosynthesis of 12-, 14- and 16-membered macrolides, Biosynthesis of secondary metabolites

MetaCyc

methymycin, neomethymycin and novamethymycin biosynthesis, narbomycin, pikromycin and novapikromycin biosynthesis

narbomycin,reduced ferredoxin:oxygen oxidoreductase (pikromycin-forming)

A cytochrome P-450 (heme-thiolate) protein. Involved in the biosynthesis of a number of bacterial macrolide antibiotics containing a desosamine glycoside unit. With narbomycin it hydroxylates at either C-12 to give pikromycin or C-14 to give neopikromycin or both positions to give narvopikromycin. With 10-deoxymethymycin it hydroxylates at either C-10 to give methymycin or C-12 to give neomethymycin or both positions to give novamethymycin.

10-deoxymethymycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

methymycin + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

5 entries

10-deoxymethymycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

neomethymycin + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

5 entries

10-deoxymethymycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

novamethymycin + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

narbomycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + 2 O2

novapikromyin + 2 oxidized ferredoxin [iron-sulfur] cluster + 2 H2O

narbomycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

neopikromycin + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

3 entries

narbomycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

novapikromycin + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

728177

Substrates: -
Products: C12,C14-dihydroxylated product of narbomycin

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narbomycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

novapikromyin + 2 oxidized ferredoxin [iron-sulfur] cluster + 2 H2O

narbomycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

pikromycin + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

5 entries

narbomycin + reduced ferredoxin [iron-sulfur] cluster + H+ + O2

pikromycin + oxidized ferredoxin [iron-sulfur] cluster + H2O

additional information

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4 entries

10-deoxymethymycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

methymycin + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

Substrates: involved in the biosynthesis of a number of bacterial macrolide antibiotics containing a desosamine glycoside unit
Products: -

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10-deoxymethymycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

neomethymycin + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

Substrates: involved in the biosynthesis of a number of bacterial macrolide antibiotics containing a desosamine glycoside unit
Products: -

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10-deoxymethymycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

novamethymycin + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

Substrates: involved in the biosynthesis of a number of bacterial macrolide antibiotics containing a desosamine glycoside unit
Products: -

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narbomycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + 2 O2

novapikromyin + 2 oxidized ferredoxin [iron-sulfur] cluster + 2 H2O

narbomycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

neopikromycin + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

Substrates: involved in the biosynthesis of a number of bacterial macrolide antibiotics containing a desosamine glycoside unit
Products: -

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narbomycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

novapikromyin + 2 oxidized ferredoxin [iron-sulfur] cluster + 2 H2O

Substrates: involved in the biosynthesis of a number of bacterial macrolide antibiotics containing a desosamine glycoside unit
Products: -

?

narbomycin + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

pikromycin + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

Substrates: involved in the biosynthesis of a number of bacterial macrolide antibiotics containing a desosamine glycoside unit
Products: -

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narbomycin + reduced ferredoxin [iron-sulfur] cluster + H+ + O2

pikromycin + oxidized ferredoxin [iron-sulfur] cluster + H2O

cytochrome P450

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ferredoxin [iron-sulfur] cluster

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heme

CYP107L2 shows a low-spin state of heme. Heme is sandwiched between helices I and L in the conserved way of P450 structures. The I-helix crosses the center of CYP107L2 in a slightly bent form over the heme structure, while helices F and G are stacked onto the I-helix to form a wide-open substrate-binding cavity just above the heme moiety

7.3

assay at

30

assay at

erythromycin C-12 hydroxylase is able to substitute for pikromycin hydroxylase which is responsible for the hydroxylation of 10-deoxymethymycin and narbomycin. Erythromycin C-12 hydroxylase accepts both 12- and 14-membered macrolides

SwissProt

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Streptomyces avermitilis ATCC 31267

UniProt

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UniProt

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725370, 727289, 727865, 728177

UniProt

Highest Expressing Human Cell Lines

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Gene Links

physiological function

3 entries

monomer

molecular modeling of the active site. For substrate narbomycin, the hydrophobic residues Ala223, Leu224, Leu225, and Leu226 may form hydrophobic interactions with the methyl group at C-6 and C-8 including C-7 of the macrolide ring

purified recombinant ligand-free CYP107L2 and its complex with lauric acid, (1) sitting drop vapor diffusion method, mixing of 500 nl of 12 mg/ml protein solution with 500 nl of reservoir solution containing 0.17 M ammonium sulfate, 0.085 M sodium cacodylate, pH 6.5, 25.5% w/v PEG 8000, and 15% v/v glycerol, for complex crystals lauric acid in a 1:10 M ratio, and equilibration against 0.05 ml of reservoir solution, 14°C, 30 days, (2) hanging drop vapor diffusion method, mixing of 0.001 ml of 12 mg/ml protein solution with 0.001 ml of reservoir solution containing 0.17 M ammonium sulfate, 0.085 M sodium cacodylate, pH 6.5, 25.5% w/v PEG 8000, and 15% v/v glycerol, and equilibration against 0.05 ml of reservoir solution, X-ray diffraction structure determination and analysis at 2.5-2.6 A resolution

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structures of the PikC D50N mutant cocrystallized with narbomycin, to 1.85 A resolution, and with 10-deoxymethymycin, to 3.2 A resolution. In the mutant D50N, the desosamine moiety of both 10-deoxymethymycin and narbomycin is bound in a catalytically productive buried site, suggesting a two-step substrate binding mechanism, whereby desosamine is recognized in the two subsites to allow the macrolide substrate to sequentially progress toward a catalytically favorable orientation

D50N

D50N/E85Q

18.4% conversion of 10-desoxymethymycin, ratio of products neomethymycin to methymycin is 0.38. 3.5% conversion of substrate narbomycin

D50N/E94Q

no conversion of 10-desoxymethymycin. 5.3% conversion of substrate narbomycin

E85A/E94A

the mutant enzyme is catalytically incompetent toward both endogenous macrolactone substrates

E85Q

E94Q

no conversion of 10-desoxymethymycin. 5.4% conversion of substrate narbomycin

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recombinant His-tagged enzyme from Escherichia coli by nickel affinity chromatography

expression in a Streptomyces venezuelae pikC mutant

expression in Escherichia coli

725370, 727289

gene cyp107l2, recombinant expression of His-tagged enzyme in Escherichia coli