Studies on the active site of deacetoxycephalosporin C synthase

Abstract

The Fe(II) and 2-oxoglutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25 % of total soluble protein in Escherichia coli and puri®ed by an ef®cient large-scale procedure. Puri®ed protein catalysed the conversions of penicillins N and G to deacetoxycephems. Gel ®ltration 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 AEFe(II) and/or 2-oxoglutarate using the hanging drop method. Crystals diffracted to beyond 1.3 A Ê resolution and belonged to the R3 space group (unit cell dimensions: a b 106.4 A Ê , c 71.2 A Ê ; a b 90 , g 120 (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 de®ned 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 ®t 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 A Ê , three nitrogen or oxygen atoms at 2.11 A Ê and one other light atom at 2.04 A Ê . For the Fe(II) in the DAOCS-2-oxoglutarate complex the EXAFS spectrum was successfully interpreted by backscattering from two His residues (Fe-N at 1.99 A Ê ), a bidentate O,O-co-ordinated 2-oxoglutarate with Fe-O distances of 2.08 A Ê , another O atom at 2.08 A Ê and one at 2.03 A Ê . 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.

MIR phasing using merohedrally twinned crystals

“…To investigate this further, a selenomethionine derivative was produced. The sequence contains six methionines and although incorporation was shown by mass spectroscopy (Lloyd et al, 1999), the MAD data collected from crystals of the SeMet protein were not readily interpretable.…”

“…Deacetoxycephalosporin C synthase (DAOCS) crystallized in space group R3 with one monomer in the asymmetric unit Lloyd et al, 1999). The crystals had a normal morphology, looked uniform under polarized light and diffracted X-rays to beyond 1.3 A Ê resolution with good scaling statistics.…”

Multiple isomorphous replacement on merohedral twins: structure determination of deacetoxycephalosporin C synthase

“…The DAOCS apo enzyme crystallizes in space group R3, with unit-cell parameters a = b = 106.4, c = 71.2 A Ê (in the hexagonal setting) and one monomer in the asymmetric unit (Valega Ê rd et al, 1998;Lloyd et al, 1999). Statistics for native and derivative data sets used in the structure determination are summarized in Table 1 (see also Valega Ê rd et al, 1998).…”

“…(v) Poor phase information from a selenomethionine derivative, even though the incorporation of the Se atoms into DAOCS had been clearly shown by mass spectroscopy (Lloyd et al, 1999) and by a¯uorescence spectrum collected on SeMet crystals.…”

“…7b). Dynamic laser lightscattering experiments on the apo enzyme (Lloyd et al, 1999) indicate that the trimeric structure is present also in solution at concentrations used in the crystallization experiments.…”