Terminally Truncated Isopenicillin N Synthase Generates a Dithioester Product: Evidence for a Thioaldehyde Intermediate during Catalysis and a New Mode of Reaction for Non-Heme Iron Oxidases.

Isopenicillin N synthase (IPNS) catalyses the four-electron oxidation of a tripeptide, l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV), to give isopenicillin N (IPN), the first-formed ß-lactam in penicillin and cephalosporin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as a co-substrate. In the absence of substrate, the carbonyl oxygen of the side-chain amide of the penultimate residue, Gln330, co-ordinates to the active-site metal iron. Substrate binding ablates the interaction between Gln330 and the metal, triggering rearrangement of seven C-terminal residues, which move to take up a conformation that extends the final α-helix and encloses ACV in the active site. Mutagenesis studies are reported, which probe the role of the C-terminal and other aspects of the substrate binding pocket in IPNS. The hydrophobic nature of amino acid side-chains around the ACV binding pocket is important in catalysis. Deletion of seven C-terminal residues exposes the active site and leads to formation of a new type of thiol oxidation product. The isolated product is shown by LC-MS and NMR analyses to be the ene-thiol tautomer of a dithioester, made up from two molecules of ACV linked between the thiol sulfur of one tripeptide and the oxidised cysteinyl ß-carbon of the other. A mechanism for its formation is proposed, supported by an X-ray crystal structure, which shows the substrate ACV bound at the active site, its cysteinyl ß-carbon exposed to attack by a second molecule of substrate, adjacent. Formation of this product constitutes a new mode of reaction for IPNS and non-heme iron oxidases in general.

Crystallographic studies on the reaction of isopenicillin N synthase with an unsaturated substrate analogue.

Isopenicillin N synthase (IPNS) catalyses conversion of the linear tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N (IPN), the central step in biosynthesis of the beta-lactam antibiotics. The unsaturated substrate analogue delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-vinylglycine (ACvG) has previously been incubated with IPNS and single product was isolated, a 2-alpha-hydroxymethyl isopenicillin N (HMPen), formed via a monooxygenase mode of reactivity. ACvG has now been crystallised with IPNS and the structure of the anaerobic IPNS:Fe(II):ACvG complex determined to 1.15 A resolution. Furthermore, by exposing the anaerobically grown crystals to high-pressure oxygen gas, a structure corresponding to the bicyclic product HMPen has been obtained at 1.60 A resolution. In light of these and other IPNS structures, and recent developments with related dioxygenases, the [2 + 2] cycloaddition mechanism for HMPen formation from ACvG has been revised, and a stepwise radical mechanism is proposed. This revised mechanism remains consistent with the observed stereospecificity of the transformation, but fits better with apparent constraints on the coordination geometry around the active site iron atom.

A device for the high-pressure oxygenation of protein crystals.

A system has been developed for subjecting protein crystals to hyperbaric pressures of oxygen gas in order to promote enzymatic reaction. Crystals of an oxygenase or oxidase enzyme are grown anaerobically by hanging drop vapor diffusion, under crystallization conditions modified to eliminate combustible materials such as plastic coverslips and grease. The crystalline enzyme:substrate complex can then be exposed to oxygen gas at pressures up to 60 bar using a custom-built device or "bomb." In this way, reaction is initiated synchronously throughout the crystal and subsequent flash freezing allows the trapping of enzyme:product complexes in high occupancy. These complexes can then be structurally characterized by conventional monochromatic X-ray crystallography. The bomb is furnished from naval brass and lubricated with Fomblin RT15 perfluorinated polyether grease in order to ensure compatibility with the highly oxidizing environment.

X-ray crystal structure of Escherichia coli taurine/alpha-ketoglutarate dioxygenase complexed to ferrous iron and substrates.

Taurine/alpha-ketoglutarate dioxygenase (TauD), a non-heme Fe(II) oxygenase, catalyses the conversion of taurine (2-aminoethanesulfonate) to sulfite and aminoacetaldehyde concurrent with the conversion of alpha-ketoglutarate (alphaKG) to succinate and CO(2). The enzyme allows Escherichia coli to use taurine, widely available in the environment, as an alternative sulfur source. Here we describe the X-ray crystal structure of TauD complexed to Fe(II) and both substrates, alphaKG and taurine. The tertiary structure and fold of TauD are similar to those observed in other enzymes from the broad family of Fe(II)/alphaKG-dependent oxygenases, with closest structural similarity to clavaminate synthase. Using the TauD coordinates, a model was determined for the closely related enzyme 2,4-dichlorophenoxyacetate/alphaKG dioxygenase (TfdA), supporting predictions derived from site-directed mutagenesis and other studies of that biodegradative protein. The TauD structure and TfdA model define the metal ligands and the positions of nearby aromatic residues that undergo post-translational modifications involving self-hydroxylation reactions. The substrate binding residues of TauD were identified and those of TfdA predicted. These results, along with sequence alignment information, reveal how TauD selects a tetrahedral substrate anion in preference to the planar carboxylate selected by TfdA, providing insight into the mechanism of enzyme catalysis.

Molybdenum and tungsten complexes of sulfene (thioformaldehyde S,S-dioxide).

Propionitrile complexes fac-[M(CO)(3)(P-P)(NCEt)] (M = Mo (3), W (4); P-P = Ph(2)PCH(2)PPh(2) (a), Ph(2)PC(2)H(4)PPh(2) (b), Ph(2)PC(3)H(6)PPh(2) (c), (S,S)-Ph(2)PCHMeCHMePPh(2) (d), Fe(C(5)H(4)PPh(2))(2) (e)) were synthesized from [M(CO)(3)(NCEt)(3)] and the corresponding diphosphine. Reactions of 3 and 4 with sulfur dioxide initially gave complexes fac-[M(CO)(3)(P-P)(eta(2)-SO(2))] (M = Mo (5), W (6)), which slowly isomerized to mer-[M(CO)(3)(P-P)(eta(1)-SO(2))] (M = Mo (7), W (8)). The structures of 7b and 8b were determined by X-ray crystallography. Both compounds are isostructural (monoclinic, space group P2(1)/n (No. 14)) with almost identical unit cell dimensions (7b, a = 14.511(5) A, b = 12.797(2) A, c = 16.476(6) A, beta = 115.92(2); 8b, a = 14.478(8) A, b = 12.794(3) A, c = 16.442(9) A, beta = 116.01(2)) and molecular geometries. Treatment of either fac-[M(CO)(3)(P-P)(eta(2)-SO(2))] or mer-[M(CO)(3)(P-P)(eta(1)-SO(2))] with diazomethane yielded the sulfene complexes mer-[M(CO)(3)(P-P)(eta(2)-CH(2)SO(2))] (M = Mo (9), W (10)). The structure of 10a was determined crystallographically: monoclinic, space group P2(1)/n (No. 14), a = 11.719(2) A, b = 17.392(4) A, c = 13.441(3) A, beta = 95.58(2). The tungsten atom resides in the center of a distorted pentagonal bipyramid. The sulfene ligand occupies two adjacent equatorial sites with the bond distances W-C, 2.322(13) A, W-S, 2.353(3) A, and S-C, 1.721(12) A. The latter equals the S-C single bond distance in thiirane S,S-dioxide, indicating a high degree of charge density transfer into the LUMO of the sulfene ligand.