3ZKP

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

Summary for 3ZKP

• Entry DOI: 10.2210/pdb3zkp/pdb
• Descriptor: ERYTHROMYCIN C-12 HYDROXYLASE, PROTOPORPHYRIN IX CONTAINING FE, Erythromycin B, ... (4 entities in total)
• Functional Keywords: oxidoreductase-antibiotic complex, catalytic domain, cytochrome p-450 enzyme system, substrate specificity, macrolide antibiotic, oxidoreductase/antibiotic
• Biological source: SACCHAROPOLYSPORA ERYTHRAEA NRRL 2338
• Total number of polymer chains: 1
• Total formula weight: 46621.33

Authors

Montemiglio, L.C.,Vallone, B.,Savino, C. (deposition date: 2013-01-24, release date: 2013-05-01, Last modification date: 2023-12-20)

Primary citation

Montemiglio, L.C.,Macone, A.,Ardiccioni, C.,Avella, G.,Vallone, B.,Savino, C.
Redirecting P450 Eryk Specificity by Rational Site-Directed Mutagenesis.
Biochemistry, 52:3678-, 2013

PubMed Abstract: 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 a byproduct decreases the yield and purity of the antibiotic. In this study, EryK has been redesigned to make it suitable for 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, and E89L) have been designed to allow hydroxylation of the nonphysiological substrate ErB. The binding and catalytic properties of these three variants on both ErD and ErB have been analyzed. 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 were recovered.

PubMed: 23597312
DOI: 10.1021/BI400223J

Experimental method

X-RAY DIFFRACTION (2 Å)