Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of C-12 hydroxylase EryK from Saccharopolyspora erythraea.
Erythromycin A is produced by Saccharopolyspora erythraea via a secondary metabolic pathway using several steps including glycosylations and hydroxylations of the first macrolide intermediate 6-deoxyerythronolide B. Erythromycin C-12 hydroxylase (CYP113A1), the P450 cytochrome active in the final stages of erythromycin biosynthesis, was cloned and expressed in E. coli. Different crystal forms were harvested from distinct crystallization conditions: two ligand-free forms, one substrate bound and two inhibitors-bound. All crystals belong either to the monoclinc P2(1)or to the orthorhombic P2(1)2(1)2(1) space groups, and exhibit diffraction limits ranging from 2.3 to 1.6 A. The structures will be determined by molecular replacement.
Savino
Carmelinda
C
Istituto di Biologia e Patologia Molecolari, CNR, and Dipartimento di Scienze Biochimiche, "Sapienza" Università di Roma, P.le A. Moro 5, I-00185 Rome, Italy. linda.savino@uniroma1.it
Sciara
Giuliano
G
Miele
Adriana E
AE
Kendrew
Steven G
SG
Vallone
Beatrice
B
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D016930:Saccharopolyspora / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System; C078882:EryK protein, Saccharopolyspora erythraea