Limited proteolysis of the cellobiohydrolase I (CBH I, 65 kDa) from Trichoderma reesei by papain yields a core protein (56 kDa) which is fully active against small, soluble substrates such as the chromophoric glycosides derived from the cellodextrins and lactose. Activity against an insoluble substrate, such as Avicel, is however completely lost and concomitantly decreased adsorption onto this microcrystalline cellulose is observed. The peptide (10 kDa), initially split off during proteolysis, is identified as the heavily glycosylated carboxy‐terminal of the native CBH I. Depending on the experimental conditions the core protein is further nicked in between disulfide bonds, but its properties and stability do not appreciably differ from those of intact CBH I. These results lead to the proposal of a bifunctional organisation of the CBH I: one domain, corresponding to the carboxyterminal, acts as a binding site for insoluble cellulose and the other, localised in the core protein, contains the active (hydrolytic) site.

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.

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