3zkp

Structure of a mutant of P450 EryK in complex with erythromycin B.

spinqualitylabelsall models 3zkp, resolution 2.00Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 3zkp is a 1 chain structure with sequence from Saccharopolyspora erythraea NRRL 2338. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2Å Ligands: ERB, HEM Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function ERYK_SACEN 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 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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