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==Structure of a mutant of P450 EryK in complex with erythromycin B.== | |||
<StructureSection load='3zkp' size='340' side='right'caption='[[3zkp]], [[Resolution|resolution]] 2.00Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[3zkp]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Saccharopolyspora_erythraea_NRRL_2338 Saccharopolyspora erythraea NRRL 2338]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3ZKP OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=3ZKP FirstGlance]. <br> | |||
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2Å</td></tr> | |||
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=ERB:ERYTHROMYCIN+B'>ERB</scene>, <scene name='pdbligand=HEM:PROTOPORPHYRIN+IX+CONTAINING+FE'>HEM</scene></td></tr> | |||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=3zkp FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3zkp OCA], [https://pdbe.org/3zkp PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=3zkp RCSB], [https://www.ebi.ac.uk/pdbsum/3zkp PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=3zkp ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[https://www.uniprot.org/uniprot/ERYK_SACEN ERYK_SACEN] | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
The C-12 hydroxylase EryK is a bacterial cytochrome P450, active during one of the final tailoring steps of erythromycin A (ErA) biosynthesis. Its tight substrate specificity, restricted to the metabolic intermediate ErD, leads to the accumulation in the culture broth of a shunt metabolite, ErB, that originates from the competitive action of a methyltranferase on the substrate of EryK. Although the methylation of the mycarosyl moiety represents the only difference between the two metabolites, EryK exhibits very low conversion of ErB in ErA via a parallel pathway. Given its limited antimicrobial activity and its moderate toxicity, contamination by such by-product decreases the yield and purity of the antibiotic. In this study, EryK has been redesigned to make it suitable to industrial application. Taking advantage of the three-dimensional structure of the enzyme in complex with ErD, three single active-site mutants of EryK (M86A, H88E, E89L) have been designed to allow hydroxylation of the non-physiological substrate ErB. The binding and catalytic properties of these three variants on both ErD and ErB have been analysed. Interestingly, we found the mutation of Met 86 to Ala to yield enzymatic activity on both ErB and ErD. The three dimensional structure of the complex of mutated EryK with ErB revealed that the mutation allows ErB to accommodate in the active site of the enzyme and to induce its closure, thus assuring the progress of the catalytic reaction. Therefore, by single mutation the fine substrate recognition, active site closure and locking was recovered. | |||
Redirecting P450 EryK specificity by rational site-directed mutagenesis.,Montemiglio LC, Macone A, Ardiccioni C, Avella G, Vallone B, Savino C Biochemistry. 2013 Apr 19. PMID:23597312<ref>PMID:23597312</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 3zkp" style="background-color:#fffaf0;"></div> | |||
== References == | |||
<references/> | |||
__TOC__ | |||
</StructureSection> | |||
[[Category: Large Structures]] | |||
[[Category: Saccharopolyspora erythraea NRRL 2338]] | |||
[[Category: Montemiglio LC]] | |||
[[Category: Savino C]] | |||
[[Category: Vallone B]] | |||