Studies on the active site of deacetoxycephalosporin C synthase

JOURNAL OF MOLECULAR BIOLOGY

卷:287

Article

Lloyd, MD ; Lee, HJ ; Harlos, K ; Zhang, ZH ; Baldwin, JE ; Schofield, CJ ; Charnock, JM ; Garner, CD ; Hara, T ; van Scheltinga, ACT ; Valegård, K ; Viklund, JAC ; Hajdu, J ; Andersson, I ; Danielsson, Å ; Bhikhabhai, R

关键词: antibiotic biosynthesis;    dioxygenase;    EXAFS;    iron;    2-oxoglutarate;   

DOI  :  10.1006/jmbi.1999.2594

来源: Elsevier

【 摘 要 】

The Fe(II) and 2-oxogolutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25% of total soluble protein in Escherichia coli and purified by an efficient large-scale procedure. Purified protein catalysed the conversions of penicillins N and G to deacetoxycephems. Gel filtration and Light scattering studies showed that in solution monomeric apo-DAOCS is in equilibrium with a trimeric form from which it crystallizes. DAOCS was crystallized +/-Fe(II) and/or 2-oxooglutarate using the hanging drop method. Crystals diffracted to beyond 1.3 Angstrom resolution and belonged to the R3 space group (unit cell dimensions: a = b = 106.4 Angstrom, c = 71.2 Angstrom; alpha = beta = 90 degrees gamma = 120 degrees (in the hexagonal setting)). Despite the structure revealing that Met180 is located close to the reactive oxidizing centre of DAOCS, there was no functional difference between the wild-type and selenomethionine derivatives. X-ray absorption spectroscopic studies in solution generally supported the iron co-ordination chemistry defined by the crystal structures. The Fe K-edge positions of 7121.2 and 7121.4 eV for DAOCS alone and with 2-oxoglutarate were both consistent with the presence of Fe(II). For Fe(II) in DAOCS the best fit to the Extended X-ray Absorption Fine Structure (EXAFS) associated with the Fe K-edge was found with two His imidazolate groups at 1.96 Angstrom, three nitrogen or oxygen atoms at 2.11 Angstrom and one other light atom at 2.04 Angstrom. For the Fe(II) in the DAOCS-2-oxoglutarate complex the EXAFS spectrum was successfully interpreted by backscattering from two His residues (Fe-nr at 1.99 Angstrom), a bidentate O,O-co-ordinated 2-oxoglutarate with Fe-O distances of 2.08 Angstrom, another O atom at 2.08 Angstrom and one at 2.03 Angstrom. Analysis of the X-ray crystal structural data suggests a binding mode for the penicillin N substrate and possible roles for the C terminus in stabilising the enzyme and ordering the reaction mechanism. (C) 1999 Academic Press.

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