The biosynthesis of penicillin and cephalosporin antibiotics in microorganisms requires the formation of the bicyclic nucleus of penicillin. Isopenicillin N synthase (IPNS), a non-haem iron-dependent oxidase, catalyses the reaction of a tripeptide, delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV), and dioxygen to form isopenicillin N and two water molecules. Mechanistic studies suggest the reaction is initiated by ligation of the substrate thiolate to the iron centre, and proceeds through an enzyme-bound monocyclic intermediate. Here we report the crystal structure of IPNS complexed to ferrous iron and ACV, determined to 1.3 A resolution. Based on the structure, we propose a mechanism for penicillin formation that involves ligation of ACV to the iron centre, creating a vacant iron coordination site into which dioxygen can bind. Subsequently, iron-dioxygen and iron-oxo species remove the requisite hydrogens from ACV without the direct assistance of protein residues. The crystal structure of the complex with the dioxygen analogue, NO and ACV bound to the active-site iron supports this hypothesis.

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The binding of penicillin to penicillin acylase was studied by X-ray crystallography. The structure of the enzyme-substrate complex was determined after soaking crystals of an inactive betaN241A penicillin acylase mutant with penicillin G. Binding of the substrate induces a conformational change, in which the side chains of alphaF146 and alphaR145 move away from the active site, which allows the enzyme to accommodate penicillin G. In the resulting structure, the beta-lactam binding site is formed by the side chains of alphaF146 and betaF71, which have van der Waals interactions with the thiazolidine ring of penicillin G and the side chain of alphaR145 that is connected to the carboxylate group of the ligand by means of hydrogen bonding via two water molecules. The backbone oxygen of betaQ23 forms a hydrogen bond with the carbonyl oxygen of the phenylacetic acid moiety through a bridging water molecule. Kinetic studies revealed that the site-directed mutants alphaF146Y, alphaF146A and alphaF146L all show significant changes in their interaction with the beta-lactam substrates as compared with the wild type. The alphaF146Y mutant had the same affinity for 6-aminopenicillanic acid as the wild-type enzyme, but was not able to synthesize penicillin G from phenylacetamide and 6-aminopenicillanic acid. The alphaF146L and alphaF146A enzymes had a 3-5-fold decreased affinity for 6-aminopenicillanic acid, but synthesized penicillin G more efficiently than the wild type. The combined results of the structural and kinetic studies show the importance of alphaF146 in the beta-lactam binding site and provide leads for engineering mutants with improved synthetic properties.

In a pilot study, the feasibility of immune whey protein concentrate (40 %; immune WPC-40) to aid the prevention of relapse of Clostridium difficile diarrhoea was evaluated. Immune WPC-40 was made from milk after immunization of Holstein-Frisian cows with C. difficile-inactivated toxins and killed whole-cell C. difficile. Immune WPC-40 contained a high concentration of specific sIgA antibodies, and was effective in neutralizing the cytotoxic effect of C. difficile toxins in cell assays in vitro. Immune WPC-40 conferred protection from otherwise lethal C. difficile-associated caecitis in hamsters. To obtain preliminary data in humans, 16 patients (10 male; median 57 years) with toxinand culture-confirmed C. difficile diarrhoea were enrolled in an uncontrolled cohort study. Nine had a history of relapsing C. difficile diarrhoea. After completion of standard antibiotic treatment, the patients received immune WPC-40 TID for 2 weeks; it was well tolerated and no treatment-related adverse effects were observed. In all but one case, C. difficile toxins had disappeared from the faeces upon completion of treatment. During a follow-up period of median 333 days (range 35 days to 1 year), none of the patients had suffered another episode of C. difficile diarrhoea. These preliminary data suggest that immune whey protein concentrate-40 may be of help in the prevention of relapse of C. difficile diarrhoea.

Desulfovibrio gigas NCIMB 9332 is a sulfate-reducing bacterium which is able to grow with H 2 , L-and D-lactate, C 4 -dicarboxylic acids, and ethanol as energy sources (17,35). D. gigas not only oxidizes ethanol (to acetate) but it can also store massive amounts of polyglucose (29) which under fermentative conditions can be degraded with ethanol as a major end product (26).Recently, we characterized the NAD-dependent alcohol dehydrogenase from D. gigas and showed it to be an oxygen-labile, decameric enzyme (14). From lactate-grown cells, a molybdenum iron-sulfur protein (MOP) which had 2,6-dichlorophenol-indophenol (DCPIP)-linked aldehyde dehydrogenase activity (1, 31) was purified. In extracts of cells grown on lactate or ethanol, high levels of activity of a coenzyme A-independent, benzylviologen-linked aldehyde dehydrogenase (BV-AlDH) were measured (17). Because at that time only the MOP was known as an enzyme with aldehyde dehydrogenase activity in D. gigas, it was suggested that the BV-AlDH activity was catalyzed by the MOP. Recently, we found that the addition of 0.1 M tungstate to the medium had a strongly stimulatory effect on the growth of D. gigas on ethanol. Omission of tungstate from the medium resulted in a decrease or absence of the BV-AlDH activity and an increase of the DCPIP-linked aldehyde dehydrogenase activity (13). These data and the different characteristics of the enzyme activities in cell extracts strongly suggested that the BV-AlDH and the MOP are two different enzymes.In this report, we describe the purification and characterization of the BV-AlDH and provide evidence that this enzyme belongs to the group of molybdopterin-and tungsten-containing aldehyde:acceptor oxidoreductases. MATERIALS AND METHODSOrganism, cultivation, and preparation of cell extract. D. gigas NCIMB 9332 was cultivated on ethanol, harvested, washed, and stored as described earlier (14), with the following minor modifications. The washing buffer was 50 mM potassium phosphate (pH 7.5) containing 2 mM dithiothreitol (DTT) and 1 mM dithionite. Cell extract was prepared by three successive passages through a French pressure cell operated at 106 MPa and centrifugation (20 min at 48,000 ϫ g and 4ЊC). The cell extract was then used for enzyme purification or stored at Ϫ20ЊC under N 2 until further use.Enzyme assays. Assay systems in cuvettes were made anaerobic by bubbling with N 2 for at least 3 min and were then closed with grey butyl rubber stoppers. The standard assay contained 50 mM potassium phosphate buffer (pH 7.5) and 2 mM benzylviologen in a total volume (after the following additions) of 1 ml. Then, 5 l of a freshly prepared dithionite solution (1.5 mM) and enzyme solution (usually 10 l) were added with microsyringes and the reaction was started by adding acetaldehyde to a final concentration of 1 mM; oxygen was removed from acetaldehyde stock solutions in crimp-seal bottles by replacing the atmosphere with oxygen-free nitrogen twice. Addition of dithionite was necessary to prevent a lag phase in the reaction. Ac...

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␣-Amino acid ester hydrolases (AEHs) catalyze the hydrolysis and synthesis of esters and amides with an ␣-amino group. As such, they can synthesize ␤-lactam antibiotics from acyl compounds and ␤-lactam nuclei obtained from the hydrolysis of natural antibiotics. This article describes the gene sequence and the 1.9-Å resolution crystal structure of the AEH from Xanthomonas citri. The enzyme consists of an ␣/␤-hydrolase fold domain, a helical cap domain, and a jellyroll ␤-domain. Structural homology was observed to the Rhodococcus cocaine esterase, indicating that both enzymes belong to the same class of bacterial hydrolases. Docking of a ␤-lactam antibiotic in the active site explains the substrate specificity, specifically the necessity of an ␣-amino group on the substrate, and explains the low specificity toward the ␤-lactam nucleus.␤-Lactam antibiotics form a large family of widely applied antibacterials. Most of them are derived from a handful of naturally occurring antibiotics like penicillin G, penicillin V, and cephalosporin C by replacing their acyl groups with synthetic ones. Initially, this was achieved by chemical means but at present, enzymatic methods are preferred (1). A well known enzyme used for these conversions is penicillin acylase (EC 3.5.1.11) from Escherichia coli. This enzyme is used both for the production of the ␤-lactam nucleus 6-aminopenicillanic acid (6-APA) 1 by cleaving off phenylacetic acid from penicillin G and for the coupling of new acyl groups to 6-APA or other ␤-lactam nuclei. Penicillin acylase is, however strongly inhibited by its product phenylacetic acid (2), which must therefore be removed before coupling of a new acyl group to the ␤-lactam nucleus can take place. In addition, ␤-lactam nuclei are not very stable at the alkaline pH optimum of penicillin acylase.By contrast, ␣-amino acid ester hydrolases (AEHs) do not have these disadvantages. These enzymes catalyze the hydrolysis and synthesis of esters and amides of ␣-amino acids exclusively, and do not attack the amide bond of a ␤-lactam. They can be used to acylate a ␤-lactam using an ester as acyl donor, as shown in Fig. 1. Because the AEHs require an ␣-amino group on the substrate, they are not inhibited by phenylacetic acid (3). Together with their ability to accept various ␤-lactam nuclei without cleaving them, this makes them suitable for generating widely used antibiotics such as ampicillin, amoxicillin, and the cephalosporins cephadroxil and cephalexin. The slightly acidic pH optimum of AEH, which is beneficial for ␤-lactam stability, is another advantage of AEHs for biocatalytic applications, as is their stereospecificity toward the acyl donor (4).One of the first AEHs that was isolated and characterized is the enzyme from Xanthomonas citri (5-11). This enzyme was found to be a homotetramer with subunits of 72 kDa (6). Kinetic studies indicated the occurrence of an acyl-enzyme intermediate in the hydrolysis and acylation reactions of ␤-lactam antibiotics (3,7,12).This article reports the gene and 1.9-Å resolution ...

Isopenicillin N synthase (IPNS) was cocrystallised with ferrous sulphate and its substrate, 6 -(~-aaminoadipoyl)-L-cysteinyl-D-valine (Aad-Cys-Val). Vital to the successful procedure was the maintenance of a rigorously anaerobic environment. Hanging-drop vapour-diffusion crystallisation experiments, using lithium sulphate as the precipitant produced three crystal forms. Form I crystals, with a plate habit, diffracted X-rays to at least 0.1 1 -nm resolution at the European Synchrotron Radiation Facility and belong to the space group P2,2,2,, with unit-cell dimensions a = 4.68, b = 7.15, c = 10.10 nm. Their asymmetric unit contains a single IPNS . Fe(I1) . Aad-Cys-Val complex with a solvent content of 38.5 %. Form I1 crystals, with a hexagonal habit, diffract X-rays to at least 0.21 nm resolution at the European Synchrotron Radiation Facility and belong to the space group P3,21, with unit-cell dimensions a = 10.10, b = 10.10, c = 11.567 nm. Their asymmetric unit also contains a single IPNS . Fe(I1) . Aad-Cys-Val complex with a solvent content of 69.5%. Form 111 crystals, needles, do not show well-ordered diffraction. Although all three forms were initially produced in crystallisation experiments under identical conditions, appropriate micro and streak seeding allows selective crystallisation of form 1 or form I1 crystals. Extended X-ray-absorption fine-structure studies on a crystalline slurry of the form I crystals demonstrate the presence of an Fe-S(Aad-Cys-Val) bond length of 0.234 2 0.003 nm.Keywords: antibiotic ; non-haem oxygenase ; penicillin biosynthesis ; isopenicillin N synthase; oxidase.The biosynthesis of penicillins occurs in a single enzyme catalysed step without synthetic precedent. Thus, isopenicillin N synthase (IPNS) catalyses the formation of both ,@lactam and thiazolidine rings during the oxidative cyclisation of ~-. The reaction has been proposed to occur via two desaturative cyclisations with concomitant transfer of four hydrogen atoms to molecular oxygen (Fig. 1). IPNS is a member of a family of ferrous-iron-dependent oxidases, many of which show structural, sequence and mechanistic similarities [2, 31. This enzyme family carries out a diverse range of biological oxidations, including desaturative cyclisations, hydroxylations and oxidative rearrangements and is involved in a wide range of metabolic processes such as the biosynthesis of antibiotics, the plant hormone ethylene and collagen.The crystal structure of catalytically inactive IPNS from Aspergillus niduluns, with Mn(1I) substituting for Fe(I1) at the active site, is thus far the only reported example of a structure from this family [3, 41. Crystals of IPNS with both the ferrous iron cofactor and the tripeptide substrate bound at the active site have proved difficult to obtain because both the substrate and cofactor are highly sensitive to oxidation. Therefore, we investigated the cocrystallisation of IPNS, Fe(I1) and Aad-Cys-Val unCorrespondence to P. L. Roach, Dyson Perrins Laboratory, South Parks Road, Oxford OX1 SQY, UK Abbreviati...