Studies on the active site of deacetoxycephalosporin C synthase.
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 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-oxoglutarate using the hanging drop method. Crystals diffracted to beyond 1.3 A resolution and belonged to the R3 space group (unit cell dimensions: a=b=106.4 A, c=71.2 A; 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 A, three nitrogen or oxygen atoms at 2.11 A and one other light atom at 2.04 A. 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), a bidentate O,O-co-ordinated 2-oxoglutarate with Fe-O distances of 2.08 A, another O atom at 2.08 A and one at 2.03 A. 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.
Lloyd
M D
MD
The Dyson Perrins Laboratory and the Oxford Centre for Molecular Sciences, South Parks Road, Oxford, OX1 3QY, UK. matthew.lloyd@chem.ox.ac.uk
Lee
H J
HJ
Harlos
K
K
Zhang
Z H
ZH
Baldwin
J E
JE
Schofield
C J
CJ
Charnock
J M
JM
Garner
C D
CD
Hara
T
T
Terwisscha van Scheltinga
A C
AC
Valegård
K
K
Viklund
J A
JA
Hajdu
J
J
Andersson
I
I
Danielsson
A
A
Bhikhabhai
R
R
D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D006860:Hydrogen Bonding; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000737:chemistry / Q000378:metabolism; D008715:Methionine; D008958:Models, Molecular; D046915:Penicillin-Binding Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013057:Spectrum Analysis / Q000379:methods; D013302:Streptomyces / Q000201:enzymology; D014965:X-Rays
D007656:Ketoglutaric Acids; D046915:Penicillin-Binding Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D008715:Methionine; D007501:Iron; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase