Publications

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

  Wu X.B., Fan K.Q., Wang Q.H., Yang K.Q.

  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 an ... >> More

  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. << Less

  FEMS Microbiol Lett 246:103-110(2005) [PubMed] [EuropePMC]

• Characterization of the cefF gene of Nocardia lactamdurans encoding a 3'-methylcephem hydroxylase different from the 7-cephem hydroxylase.

  Coque J., Enguita F.J., Cardoza R.E., Martin J.F., Liras P.

  The cefF gene of Nocardia lactamdurans, encoding a functional 2-oxoglutarate-dependent 3'-methylcephem hydroxylase (deacetoxycephalosporin C hydroxylase) has been found to be closely linked to the pcbC gene in the cephamycin C gene cluster. The open-reading frame is 933 bp long and could encode a ... >> More

  The cefF gene of Nocardia lactamdurans, encoding a functional 2-oxoglutarate-dependent 3'-methylcephem hydroxylase (deacetoxycephalosporin C hydroxylase) has been found to be closely linked to the pcbC gene in the cephamycin C gene cluster. The open-reading frame is 933 bp long and could encode a protein of M(r) 34,366. Introduction of cefF in the cephamycin-non-producer Streptomyces lividans conferred 3'-methylcephem-hydroxylating activity to the transformants but did not result in hydroxylation at carbon 7 of cephamycin. No 3'-methylcephem hydroxylase activity was observed when the cefF gene was introduced in S. lividans in pIJ699 (a vector containing two transcriptional terminators that prevent read-through expression), which suggests that this gene lacks an independent promoter. << Less

  Appl. Microbiol. Biotechnol. 44:605-609(1996) [PubMed] [EuropePMC]

• Refolding and purification of Cephalosporium acremonium deacetoxycephalosporin C synthetase/hydroxylase from granules of recombinant Escherichia coli.

  Ghag S.K., Brems D.N., Hassell T.C., Yeh W.K.

  The gene for bifunctional deacetoxycephalosporin C synthetase/hydroxylase of Cephalosporium acremonium was cloned and overexpressed as an insoluble and inactive enzyme in granules of recombinant Escherichia coli. About 40-60% of expected synthetase activity along with 50-80% protein purity could b ... >> More

  The gene for bifunctional deacetoxycephalosporin C synthetase/hydroxylase of Cephalosporium acremonium was cloned and overexpressed as an insoluble and inactive enzyme in granules of recombinant Escherichia coli. About 40-60% of expected synthetase activity along with 50-80% protein purity could be recovered directly from granular extracts with only a single empirically optimized refolding step. Further purification to homogeneity was achieved by a single anion-exchange-chromatographic step in the presence of denaturing concentrations of urea. The main obstacle to converting the homogeneous unfolded protein into the active enzyme was a urea-dependent aggregation during refolding that led to irreversible enzyme inactivation. Information obtained from refolding studies using gel-filtration HPLC, fluorescence spectroscopy and disulphide analysis led to an optimal enzyme refolding scheme that resulted in a highly active (i.e. 65-75% of the expected activity) and moderately stable fungal synthetase/hydroxylase. << Less

  Biotechnol Appl Biochem 24:109-119(1996) [PubMed] [EuropePMC]

• Expression of genes and processing of enzymes for the biosynthesis of penicillins and cephalosporins.

  Martin J.F., Gutierrez S., Fernandez F.J., Velasco J., Fierro F., Marcos A.T., Kosalkova K.

  The genes pcbAB, pcbC and penDE encoding the enzymes (alpha-aminoadipyl-cysteinyl-valine synthetase, isopenicillin N synthase and isopenicillin N acyltransferase, respectively) involved in the biosynthesis of penicillin have been cloned from Penicillin chrysogenum and Aspergillus nidulans. They ar ... >> More

  The genes pcbAB, pcbC and penDE encoding the enzymes (alpha-aminoadipyl-cysteinyl-valine synthetase, isopenicillin N synthase and isopenicillin N acyltransferase, respectively) involved in the biosynthesis of penicillin have been cloned from Penicillin chrysogenum and Aspergillus nidulans. They are clustered in chromosome I (10.4 Mb) of P. chrysogenum, in chromosome II of Penicillium notatum (9.6 Mb) and in chromosome VI (3.0 Mb) of A. nidulans. Each gene is expressed as a single transcript from separate promoters. Enzyme regulation studies and gene expression analysis have provided useful information to understand the control of genes involved in penicillin biosynthesis. The enzyme isopenicillin N acyltransferase encoded by the penDE gene is synthesized as a 40 kDa protein that is (self)processed into two subunits of 29 and 11 kDa. Both subunits appear to be required for acyl-CoA 6-APA acyltransferase activity. The isopenicillin N acyltransferase was shown to be located in microbodies, whereas the isopenicillin N synthase has been reported to be present in vesicles of the Golgi body and in the cell wall. A mutant in the carboxyl-terminal region of the isopenicillin N acyltransferase lacking the three final amino acids of the enzymes was not properly located in the microbodies and failed to synthesize penicillin in vivo. In C. acremonium the genes involved in cephalosporin biosynthesis are separated in at least two clusters. Cluster I (pcbAB-pcbC) encodes the first two enzymes (alpha-aminoadipyl-cysteinyl) valine synthetase and isopenicillin N synthase) of the cephalosporin pathway which are very similar to those involved in penicillin biosynthesis. Cluster II (cefEF-cefG), encodes the last three enzymatic activities (deacetoxycephalosporin C synthetase/hydroxylase and deacetylcephalosporin C acetyltransferase) of the cephalosporin pathway. It is unknown, at this time, if the cefD gene encoding isopenicillin epimerase is linked to any of these two clusters. Methionine stimulates cephalosporin biosynthesis in cultures of three different strains of A. chrysogenum. Methionine increases the levels of enzymes (isopenicillin N synthase and deacetylcephalosporin C acetyltransferase) expressed from genes (pcbC and cefG respectively) which are separated in the two different clusters of cephalosporin biosynthesis genes. This result suggests that both clusters of genes have regulatory elements which are activated by methionine. Methionine-supplemented cells showed higher levels of transcripts of the pcbAB, pcbC, cefEF genes and to a lesser extent of cefG than cells grown in absence of methionine. The levels of the cefG transcript were very low as compared to those of pcbAB, pcbC and cefEF.(ABSTRACT TRUNCATED AT 400 WORDS) << Less

  Antonie Van Leeuwenhoek 65:227-243(1994) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

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

  Baker B.J., Dotzlaf J.E., Yeh W.K.

  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 35,000-3 ... >> More

  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. << Less

  J. Biol. Chem. 266:5087-5093(1991) [PubMed] [EuropePMC]

• Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase.

  Lloyd M.D., Lipscomb S.J., Hewitson K.S., Hensgens C.M., Baldwin J.E., Schofield C.J.

  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 deacetoxycephalo ... >> More

  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. << Less

  J Biol Chem 279:15420-15426(2004) [PubMed] [EuropePMC]