Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase

Deacetoxycephalosporin/deacetylcephalosporin C synthase (DAOC/DACS) is an iron(II) and 2-oxoglutarate-dependent oxygenase involved in the biosynthesis of cephalosporin C in Cephalosporium acremonium. It catalyzes two oxidative reactions, oxidative ring-expansion of penicillin N to deacetoxycephalosporin C, and hydroxylation of the latter to give deacetylcephalosporin C. The enzyme is closely related to deacetoxycephalosporin C synthase (DAOCS) and DACS from Streptomyces clavuligerus, which selectively catalyze ring-expansion or hydroxylation reactions, respectively. In this study, structural models based on DAOCS coupled with site-directed mutagenesis were used to identify residues within DAOC/DACS that are responsible for controlling substrate and reaction selectivity. The M306I mutation abolished hydroxylation of deacetylcephalosporin C, whereas the W82A mutant reduced ring-expansion of penicillin G (an "unnatural" substrate). Truncation of the C terminus of DAOC/DACS to residue 310 (Delta310 mutant) enhanced ring-expansion of penicillin G by approximately 2-fold. A double mutant, Delta310/M306I, selectively catalyzed the ring-expansion reaction and had similar kinetic parameters to the wild-type DAOC/DACS. The Delta310/N305L/M306I triple mutant selectively catalyzed ring-expansion of penicillin G and had improved kinetic parameters (K(m) = 2.00 +/- 0.47 compared with 6.02 +/- 0.97 mm for the wild-type enzyme). This work demonstrates that a single amino acid residue side chain within the DAOC/DACS active site can control whether the enzyme catalyzes ring-expansion, hydroxylation, or both reactions. The catalytic efficiency of mutant enzymes can be improved by combining active site mutations with other modifications including C-terminal truncation and modification of Asn-305.     

Deacetoxycephalosporin C synthase isozymes exhibit diverse catalytic activity and substrate specificity

The biosynthesis of cephalosporins involving a thiozolidine ring expansion is catalyzed by deacetoxycephalosporin C synthase (DAOCS). In this study, three DAOCS isozymes were cloned and expressed as active enzymes together with Streptomyces jumonjinensis DAOCS that was newly isolated and partially characterized. The enzymes showed excellent substrate conversion for penicillin G, phenethicillin, ampicillin and carbenicillin, but they were less effective in the ring expansion of penicillin V, amoxicillin and metampicillin. Streptomyces clavuligerus DAOCS was the most active among the recombinant enzymes. The results also showed that truncation of 20 amino acids at the C-terminus of the Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase polypeptide did not affect penicillin ring expansion.     

Molecular analysis of the gene cluster involved in cephalosporin biosynthesis from Lysobacter lactamgenus YK90

Determination of the nucleotide sequence downstream from the Lysobacter lactamgenus pcbC gene encoding isopenicillin N synthase revealed that five open-reading frames (ORF) including the pcbC gene were tightly linked in the same orientation. Each ORF has the remarkable feature of the protein-coding frame in the DNA sequence with a high G + C content. Expression in Escherichia coli and a comparison of the deduced amino acid sequences with published sequences showed that the gene cluster contained a deacetoxycephalosporin C synthetase (DAOCS) gene (cefE), an ORF having homology with the Cephalosporium acremonium DAOCS/deacetylcephalosporin C synthetase gene (cefEF), an isopenicillin N epimerase gene(cefD), and a -lactamase gene. The gene order was pcbC-cefE-ORF3-cefD--lactamase.     

Deacetoxycephalosporin C hydroxylase of Streptomyces clavuligerus. Purification, characterization, bifunctionality, and evolutionary implication

Deacetoxycephalosporin C hydroxylase from cell-free extracts of Streptomyces clavuligerus was stabilized partially and purified to near homogeneity by three anion-exchange chromatographies, ammonium sulfate fractionation, and two gel filtrations. The hydroxylase was a monomer with a Mr of 35,000-38,000. alpha-Ketoglutarate, ferrous iron, and molecular oxygen were required for the enzyme activity. The hydroxylase was optimally active between pH 7.0 and 7.4 in a 3-(N-morpholino)propanesulfonic acid buffer and at 29 degrees C. It was stimulated by a reducing agent, particularly dithiothreitol or reduced glutathione, and ATP. The requirement for ferrous ion was specific, and at least one sulfhydryl group was apparently essential for the enzymatic hydroxylation. The Km values of the hydroxylase for deacetoxycephalosporin C and alpha-ketoglutarate were 59 and 10 microM, respectively, and the Ka for ferrous ion was 20 microM. In addition to its known hydroxylation of deacetoxycephalosporin C to deacetylcephalosporin C, the hydroxylase catalyzed effectively an analogous hydroxylation of 3-exomethylenecephalosporin C to deacetoxycephalosporin C. Surprisingly, the hydroxylase also mediated slightly a novel ring-expansion of penicillin N to deacetoxycephalosporin C. The substrate specificity of the hydroxylase is overlapping with but distinguishable from that of deacetoxycephalosporin C synthase, the enzyme which normally mediates the ring-expansion reaction (Dotzlaf, J. E., and Yeh, W. K. (1989) J. Biol. Chem. 264, 10219-10227). Furthermore, the hydroxylase exhibited an extensive sequence similarity to the synthase. Thus, the two enzymes catalyzing the consecutive reactions for cephamycin C biosynthesis in S. clavuligerus represent apparent products from a divergent evolution.     

Oxygen derepresses deacetoxycephalosporin C synthase and increases the conversion of penicillin N to cephamycin C in Streptomyces clavuligerus.

When dissolved oxygen (DO) was maintained at saturation level during batch fermentations of Streptomyces clavuligerus (NRRL 3585), the accumulation of the intermediate penicillin N was lowered while formation of the end product cephamycin C was increased relative to fermentations without DO control. The specific activity of the penicillin ring-expansion enzyme deacetoxycephalosporin C synthase (DAOCS) was increased 2.3-fold under oxygen saturated conditions, whereas the penicillin ring-cyclizing enzyme isopenicillin N synthase (IPNS) showed only a 1.3-fold increase. Thus oxygen derepression of DAOCS appears to be an important regulatory mechanism in the conversion of penicillin N to cephamycin C in S. clavuligerus. IPNS, an early acting enzyme in cephamycin C biosynthesis, and DAOCS, which acts late in the pathway, both disappeared from cell extracts at 60 h, just prior to cessation of cephamycin production.     

C-terminus mutations of Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase with improved activity toward penicillin analogs

Deacetoxy/deacetylcephalosporin C synthase (acDAOC/DACS) from Acremonium chrysogenum is a bifunctional enzyme that catalyzes both the ring-expansion of penicillin N to deacetoxycephalosporin C (DAOC) and the hydroxylation of the latter to deacetylcephalosporin C (DAC). Three residues N305, R307 and R308 located in close proximity to the C-terminus of acDAOC/DACS were each mutated to leucine. The N305L and R308L mutant acDAOC/DACSs showed significant improvement in their ability to convert penicillin analogs. R308 was identified for the first time as a critical residue for DAOC/DACS activity. Kinetic analyses of purified R308L enzyme indicated its improved catalytic efficiency is due to combined improvements of K(m) and k(cat). Comparative modeling of acDAOC/DACS supports the involvement of R308 in the formation of substrate-binding pocket.     

Saturation mutagenesis of Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase R308 site confirms its role in controlling substrate specificity

Deacetoxy/deacetylcephalosporin C synthase (acDAOC/DACS) from Acremonium chrysogenum is a bifunctional enzyme that catalyzes both the ring-expansion of penicillin N to deacetoxycephalosporin C and the hydroxylation of the latter to deacetylcephalosporin C. The R308 residue located in close proximity to the C-terminus of acDAOC/DACS was mutated to the other 19 amino acids. In the resulting mutant pool, R308L, R308I, R308T and R308V showed significant improvement in their ability to convert penicillin analogs, thus confirming the role of R308 in controlling substrate selectivity, the four amino acids all possess short aliphatic sidechains that may improve hydrophobic interactions with the substrates. The mutant R308I showed the highest reactivity for penicillin G, with 3-fold increase in k_cat/K_m ratio and 7-fold increase in relative activity.     

Purification and properties of deacetoxycephalosporin C synthase from recombinant Escherichia coli and its comparison with the native enzyme purified from Streptomyces clavuligerus

A putatively rate-limiting synthase (expandase) of Streptomyces clavuligerus was stabilized in vitro and purified 46-fold from cell-free extracts; a major enriched protein with a Mr of 35,000 was further purified by electrophoretic elution. Based on a 22-residue amino-terminal sequence of the protein, the synthase gene of S. clavuligerus was cloned and expressed in Escherichia coli (Kovacevic, S., Weigel, B.J., Tobin, M.B., Ingolia, T.D., and Miller, J. R. (1989) J. Bacteriol. 171, 754-760). The synthase protein was detected mainly from granules of recombinant E. coli. The recombinant synthase was solubilized from the granules by urea, and for the first time a highly active synthase was purified to near homogeneity. The synthase was a monomer with a Mr of 34,600 and exhibited two isoelectric points of 6.1 and 5.3. Its catalytic activity required alpha-ketoglutarate, Fe2+, and O2, was stimulated by dithiothreitol or ascorbate but not by ATP, and was optimal at pH 7.0 in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer and at 36 degrees C. The Fe2+ requirement was specific, and at least one sulfhydryl group in the purified enzyme was apparently essential for the ring expansion. The Km values of the enzyme for penicillin N and alpha-ketoglutarate were 29 and 18 microM, respectively, and the Ka for Fe2+ was 8 microM. The recombinant synthase was indistinguishable from the native synthase of S. clavuligerus by those biochemical properties. In addition to the enzymic ring expansion of penicillin N to deacetoxycephalosporin C, the recombinant synthase catalyzed a novel hydroxylation of 3-exomethylenecephalosporin C to deacetylcephalosporin C.     

Cloning and location of a gene governing lysine epsilon-aminotransferase, an enzyme initiating beta-lactam biosynthesis in Streptomyces spp.

In actinomycetes that produce beta-lactam antibiotics of the cephem type, lysine epsilon-aminotransferase is the initial enzyme in the conversion of lysine to alpha-aminoadipic acid. We used a two-stage process ("chromosome walking") to screen a lambda library of Streptomyces clavuligerus genomic DNA for fragments that expressed lysine epsilon-aminotransferase activity in S. lividans. Restriction analysis of the cloned DNA confirmed the location of the putative lat gene within the cluster of beta-lactam biosynthesis genes, roughly midway between pcbC, the structural gene for isopenicillin N synthetase, and the putative cefE gene encoding deacetoxycephalosporin C synthetase.     

Extracellular production of biologically active deacetoxycephalosporin C synthase from Streptomyces clavuligerus in Pichia pastoris.

We have successfully expressed and observed secretion of the Streptomyces clavuligerus deacetoxycephalosporin C synthase (DAOCS) using the Pichia pastoris expression system. Two clones having multiple copies of the expression cassette were selected and used for protein-expression analysis. SDS-PAGE showed efficient expression and secretion of the bacterial recombinant DAOCS. The highest yield (120 microg/mL) was obtained when expression was induced with 2% methanol. Free and immobilized protein were assayed for biological activity and found to expand penicillin N (its natural substrate) and penicillin G to deacetoxycephalosporin C (DAOC) and deacetoxycephalosporin G (DAOG), respectively.     

Engineering Streptomyces clavuligerus deacetoxycephalosporin C synthase for optimal ring expansion activity toward penicillin G

The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application. A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, V275I, and C281Y, that showed a two- to sixfold increase in the k(cat)/K(m) ratio compared to the wild-type enzyme. Site-directed mutagenesis to modify residues surrounding the substrate resulted in three mutants, N304K, I305L, and I305M, with 6- to 14-fold-increased k(cat)/K(m) values. When mutants containing all possible combinations of these six sites were generated to optimize the ring expansion activity for penicillin G, the double mutant, YS67 (V275I, I305M), showed a significant 32-fold increase in the k(cat)/K(m) ratio and a 5-fold increase in relative activity for penicillin G, while the triple mutant, YS81 (V275I, C281Y, I305M), showed an even greater 13-fold increase in relative activity toward penicillin G. Our results demonstrate that this is a robust approach to the modification of DAOCS for an optimized DAOCS-penicillin G reaction.     

Mutational evidence supporting the involvement of tripartite residues His183, Asp185, and His243 in Streptomyces clavuligerus deacetoxycephalosporin C synthase for catalysis

Deacetoxycephalosporin C synthase (DAOCS) is a non-heme iron-binding and alpha-ketoglutarate dependent enzyme involved in catalyzing the biosynthesis of cephalosporins and cephamycins, antibiotics more potent than penicillins. In the crystal structure complex of Streptomyces clavuligerus DAOCS (scDAOCS), it was proposed that histidine-183, aspartate-185, and histidine-243 are putative iron-binding ligands. However, coordinates proposed for crystal structures of proteins may not definitely comply with catalysis. Hence, site-directed mutagenesis was done to replace each of these amino acid residues with leucine. The constructed expression vectors bearing the mutations were found to express the respective scDAOCS mutant enzymes at high levels in Escherichia coli BL21(DE3). Through enzymatic assays, it was shown that while the wildtype enzyme could convert penicillin to a more active cephalosporin, the substitution of the three proposed iron-binding sites of scDAOCS completely abolished the same activity in the respective mutant enzymes. Thus, these results clearly indicate that histidine-183, aspartate-185, and histidine-243 of scDAOCS are essential for the ring expansion activity.     

Cloning and sequencing of the beta-lactam hydroxylase gene (cefF) from Streptomyces clavuligerus: gene duplication may have led to separate hydroxylase and expandase activities in the actinomycetes.

The deacetylcephalosporin C synthetase (hydroxylase) gene from Streptomyces clavuligerus has been cloned and sequenced. The open reading frame codes for a protein with an Mr of 34,584. The hydroxylase gene (cefF) is closely linked to the epimerase gene (cefD) and the expandase gene (cefE) and is transcribed in the opposite orientation. The hydroxylase and expandase genes are 59 and 71% identical at the amino acid and DNA levels, respectively. cefE and cefF may have arisen from a gene duplication in the actinomycetes.     

Mutation of N304 to leucine in Streptomyces clavuligerus deacetoxycephalosporin C synthase creates an enzyme with increased penicillin analogue conversion

Superimposition of deacetoxycephalosporin C synthase (DAOCS) and isopenicillin N synthase (IPNS) structures revealed that R74, R160, R266 and N304 are strategically located in the catalytic cavity of Streptomyces clavuligerus DAOCS (scDAOCS) and are crucial for orchestrating different substrates. Substitutions at these sites to a hydrophobic leucine residue were expected to stabilize the hydrophobic substrate bound state. Substantial improvements in the biotransformation of penicillin G, ampicillin and amoxicillin to their respective cephalosporin moieties were observed using the N304L mutant scDAOCS. Thus, our results have demonstrated the enhancement of scDAOCS activity via critical computational analysis and site-directed mutagenesis of endogenous ligands.     

Cloning and nucleotide sequence determination of the isopenicillin N synthetase gene from Streptomyces clavuligerus

The isopenicillin N synthetase (IPNS) gene from Streptomyces clavuligerus was isolated from an Escherichia coli plasmid library of S. clavuligerus genomic DNA fragments using a 44-mer mixed oligodeoxynucleotide probe. The nucleotide sequence of a 3-kb region of the cloned fragment from the plasmid, pBL1, was determined and analysis of the sequence showed an open reading frame that could encode a protein of 329 amino acids with an Mr of 36,917. When the S. clavuligerus DNA from pBL1 was introduced into an IPNS-deficient mutant of S. clavuligerus on the Streptomyces vector pIJ941, the recombinant plasmid was able to complement the mutation and restore IPNS activity. The protein coding region of the S. clavuligerus IPNS gene shows about 63% and 62% similarity to the Cephalosporium acremonium and Penicillium chrysogenum IPNS nucleotide sequences, respectively, and the predicted amino acid sequence of the encoded protein showed about 56% similarity to both fungal sequences.     

The conversion of cephalosporins to 7 alpha-methoxycephalosporins by cell-free extracts of Streptomyces clavuligerus.

In the presence of S-adenosylmethionine, 2-oxoglutarate, Fe2+ and a reducing agent, cell-free extracts of Streptomyces clavuligerus convert cephalosporin C and O-carbamoyldeacetylcephalosporin C into 7 alpha-methoxy derivatives. No synthesis of a 7 alpha-methoxy derivative of deacetylcephalosporin C was detected in the system used, and the 7 alpha-methoxy derivative of deacetoxycephalosporin C was produced only in relatively small amounts. It appears that the 7 alpha-methoxy group is introduced after the cephalosporin ring system has been formed and that its introduction may represent the final step in a biosynthetic pathway.     

Improvement in the bioconversion of penicillin G to deacetoxycephalosporin G by elimination of agitation and addition of decane

The bioconversion of penicillin G to deacetoxycephalosporin G (DAOG) using resting cells of Streptomyces clavuligerus could be a very valuable step in the economical production of semisynthetic cephalosporin antibiotics. The extent of the reaction, however, is very low due to inactivation of the ring expansion enzyme deacetoxycephalosporin C synthetase ("expandase") by reaction components. We show that elimination of agitation during the reaction lowers the rate but increases the amount of DAOG produced, presumably because the inactivation requires high levels of oxygen. Many additives to the medium were examined for their effect on the reaction. Clearly, the most effective compound was the organic solvent, decane.     

Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities.

Deacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40,000) exhibited a pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+ and O2 as cofactors, and ascorbate and dithiothreitol were necessary for maximum activity. It was stable for over 4 weeks at -70 degrees C in the presence of 1 mM-dithiothreitol. Activity was inhibited by the thiol-quenching reagent N-ethylmaleimide, the metal-ion-chelating reagent bathophenanthroline, and NH4HCO3. The highly purified enzyme also showed deacetoxycephalosporin C hydroxylase (deacetylcephalosporin C synthetase) activity, indicating that both expandase and hydroxylase activities are properties of a single protein. These activities could not be separated by ion-exchange, dye-ligand, gel-filtration or hydrophobic chromatography. A beta-sulphoxide and a 3 beta-methylene hydroxy analogue of penicillin N were synthesized to test as potential intermediates in the ring-expansion reaction, Neither compound was a substrate for the enzyme. A synthetic analogue in which the 3 beta-methyl group and the 2-hydrogen atom of penicillin N were replaced by a cyclopropane ring was not a substrate but was a reversible inhibitor of the enzyme.     

Purification and initial characterization of deacetoxycephalosporin C synthase from Streptomyces clavuligerus

Deacetoxycephalosporin C synthase, the penicillin N ring expansion enzyme from Streptomyces clavuligerus, was purified to near homogeneity, as judged by sodium dodecyl sulphate - polyacrylamide gel electrophoresis. The synthase was monofunctional and could be completely separated from deacetoxycephalosporin C hydroxylase activity early in the purification sequence. Synthase specific activity was increased 97-fold over crude cell-free extracts, and the purified enzyme appeared to be a monomer with a molecular weight of 36,000 and a Km for the penicillin N substrate of 50 microM. Deacetoxycephalosporin C synthase activity required alpha-ketoglutarate, Fe2+, and oxygen and was specifically stimulated by ascorbate and dithiothreitol. The enzyme was sensitive to thiol-specific inhibitors, the most effective of which was N-ethylmaleimide.