Cloning and characterization of femA and femB from Staphylococcus epidermidis.

A DNA fragment was identified and cloned from Staphylococcus epidermidis (Se) using femA from S. aureus (Sa) as a heterologous hybridization probe. DNA sequence analysis of a portion of this clone revealed two complete ORFs highly related to femA and femB of Sa. The genomic arrangement of the Se femA/B complex was nearly identical to that observed in Sa. Intra- and interspecies relatedness of these genes and conservation of genomic organization were consistent with gene duplication of one of these genes in an ancestral organism. Recombinant FEMA, produced in Escherichia coli (Ec), was purified to near homogeneity. Identity of the purified protein was verified by N-terminal amino acid (aa) sequence analysis.

Improved expression of a hybrid Streptomyces clavuligerus cefE gene in Penicillium chrysogenum.

A hybrid cefE gene, encoding penicillin N expandase, was constructed by fusing the promoter sequences, Pcp, and terminator sequences, Pct from the Penicillium chrysogenum pcbC gene to the open reading frame (orf), cefEorf, from the Streptomyces clavuligerus cefE gene. The resulting hybrid gene, Pcp/cefE'orf/Pct, differed from a previously reported hybrid cefE gene contained on plasmid pPS65. The latter gene, Pcp/cefE'orf/Sct, contained the Pcp sequences fused to the S. clavuligerus cefE orf still attached to the S. clavuligerus terminator sequences, Sct. The new hybrid gene was transformed into P. chrysogenum on plasmid vector pRH6. Transformants were selected by phleomycin resistance conferred by a hybrid ble gene present on plasmid pRH6. The hybrid ble gene was formed by attaching Pcp sequences to the ble orf. Among transformants obtained with pRH6, one exhibited a 70-fold higher level of activity of penicillin N expandase than the best transformant previously obtained from a 10-fold larger population of pPS65 transformants. The penicillin N expandase activity in pRH6 transformant, 9EN-5-1, was fourfold higher than the activity in the S. clavuligerus strain used as the source of the cefE orf and 75% of the activity observed in an industrial strain of Cephalosporium acremonium. Sequencing of the junctions of the heterologous DNA in Pcp/cefEorf/Pct uncovered a modification of the cefE open reading frame introduced during construction of the hybrid gene; the modified open reading frame is designated cefE'orf.

Evolving enzyme technology for pharmaceutical applications: case studies.

The case studies focus on two types of enzyme applications for pharmaceutical development. Demethylmacrocin O-methyltransferase, macrocin O-methyltransferase (both putatively rate-limiting) and tylosin reductase were purified from Streptomyces fradiae, characterized and the genes manipulated for increasing tylosin biosynthesis in S. fradiae. The rate-limiting enzyme, deacetoxycephalosporin C (DAOC) synthase/hydroxylase (expandase/ hydroxylase), was purified from Cephalosporium acremonium, its gene over-expressed, and cephalosporin C biosynthesis improved in C. acremonium. Also, heterologous expression of penicillin N epimerase and DAOC synthase (expandase) genes of Streptomyces clavuligerus in Penicillium chrysogenum permitted DAOC production in the fungal strain. Second, serine hydroxymethyltransferase of Escherichia coli and phthalyl amidase of Xanthobacter agilis were employed in chemo-enzymatic synthesis of carbacephem. Similarly, echinocandin B deacylase of Actinoplanes utahensis was used in the second-type synthesis of the ECB antifungal agent.

Origins of metabolic diversity: substitution of homologous sequences into genes for enzymes with different catalytic activities.

Similar amino acid sequences were found in portions of bacterial enzymes that mediate different biochemical transformations. Reaction catalyzed by the enzymes include oxygenation, decarboxylation, isomerization, and hydrolysis. The proteins share a common evolutionary history because they participate in an overall catabolic process known as the beta-ketoadipate pathway. One interpretation of the sequence similarities might be that duplication of a single gene gave rise to ancestral genes for the enzymes with different catalytic activities. According to this view, homologous sequences from the ancestral gene were conserved as the proteins diverged to assume different functions. This hypothesis is vitiated by comparison of the NH2-terminal amino acid sequences of sets of enzymes that mediate identical or analogous metabolic reactions within an organism. Gene duplications giving rise to the enzymes within each set must have followed duplication of a putative ancestral gene for all the sets. Yet the amino acid sequences of the proteins within each set have diverged widely, and against this background of divergence the conservation of sequences from an ancestor common to all the enzymes is unlikely. Rather, it appears that most regions of sequence similarity shared by enzymes from different sets were acquired subsequent to their divergence from any common ancestor. In some cases it appears that relatively short regions of sequence homology were achieved by mutations causing the transfer of sequence information from one set of structural genes to structural genes in another set. Alignment of homologous amino acid sequences within any single set requires the introduction of few gaps. Because gaps are required to align sequences that have been altered by the insertion of genetic material, the evidence indicates that copies of oligonucleotides were exchanged by genetic substitution among different structural genes as they coevolved.

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.

Copurification and characterization of deacetoxycephalosporin C synthetase/hydroxylase from Cephalosporium acremonium.

Deacetoxycephalosporin C synthetase (expandase), which catalyzes ring expansion of penicillin N to deacetoxycephalosporin C (DAOC), has been stabilized in vitro and purified to near homogeneity from the industrially important fungus Cephalosporium acremonium. Throughout the purification, the expandase activity remained physically associated with and in a constant ratio of 7:1 to DAOC hydroxylase activity. The latter activity mediates hydroxylation of DAOC to deacetylcephalosporin C (DAC). The copurified expandase/hydroxylase appeared to be monomeric, with a molecular weight of 41,000 +/- 2,000 and an isoelectric point of 6.3 +/- 0.3. Both catalytic activities required alpha-ketoglutarate, Fe2+, and O2 and were stimulated by ascorbate, dithiothreitol, and ATP. The Fe2+ requirement was specific, and sulfhydryl groups in the purified protein were apparently essential for both ring expansion and hydroxylation. The kinetics and stoichiometry of DAOC/DAC formation from the expandase/hydroxylase-catalyzed reactions suggested that ring expansion of penicillin N preceded hydroxylation of DAOC.

Similar structures in gamma-carboxymuconolactone decarboxylase and beta-ketoadipate succinyl coenzyme A transferase.

gamma-Carboxymuconolactone decarboxylase (EC 4.1.1.44) and beta-ketoadipate succinyl coenzyme A transferase (EC 2.8.3.6) mediate different steps in the beta-ketoadipate pathway. Antisera prepared against the Pseudomonas putida transferase cross-reacted immunologically with the decarboxylase from the same organism. The transferase is formed by association of two nonidentical protein subunits. The NH2-terminal amino acid sequences of the two nonidentical transferase subunits resembled each other and also were similar to the NH2-terminal amino acid sequence of the decarboxylase.

Evolutionarily homologous alpha 2 beta 2 oligomeric structures in beta-ketoadipate succinyl-CoA transferases from Acinetobacter calcoaceticus and Pseudomonas putida.

Homogeneous beta-ketoadipate succinyl-CoA transferase (EC 2.8.3.6) preparations were obtained from extracts of Acinetobacter calcoaceticus and Pseudomonas putida. Gel filtration indicated that the respective transferases have similar molecular weights of 108,000 and 109,000; each transferase appears to have an alpha 2 beta 2 oligomeric structure formed by association of nonidentical subunits with a molecular weight of about 25,000. The subunits were separated by sodium dodecyl sulfate-gel electrophoresis, and differences in their primary structures were revealed by determination of the NH2-terminal amino acid sequences of the oligomers. The transferases cross-react immunologically and possess similar amino acid compositions. These are remarkably similar to the amino acid compositions of gamma-carboxymuconolactone decarboxylases (EC 4.1.1.44) and beta-ketoadipate enol-lactone hydrolases (EC 3.1.1.24), enzymes that mediate consecutive reactions preceding the transferase step in the beta-ketoadipate pathway.

Origins of metabolic diversity: evolutionary divergence by sequence repetition.

Recurring patterns of primary structure have been observed in enzymes that mediate sequential metabolic reactions in bacteria. The enzymes, muconolactone Delta-isomerase [(+)-4-hydroxy-4-carboxymethylisocrotonolactone Delta(2)-Delta(3)-isomerase, EC 5.3.3.4] and beta-ketoadipate enol-lactone hydrolase [4-carboxymethylbut-3-enolide(1,4)enol-lactone-hydrolase, EC 3.1.1.24], have been coselected in bacterial populations because the isomerase can confer no nutritional advantage in the absence of the hydrolase. Similar amino acid sequences recur within the structure of the isomerase, and the amino-terminal amino acid sequence of the isomerase from Pseudomonas putida appears to be evolutionarily homologous with the corresponding sequence of a beta-ketoadipate enol-lactone hydrolase from Acinetobacter calcoaceticus. One interpretation of the sequence repetitions is that they reflect tandem duplication mutations that took place early in the evolution of the proteins. According to this view, the mutations caused elongation of structural genes and the creation of duplicated genes as the metabolic pathways evolved. A review of the sequence data calls attention to a different hypothesis: repeated amino acid sequences were introduced in the course of the proteins' evolution by substitution of copies of DNA sequences into structural genes. Our observations are interpreted on the basis of a model proposing genetic exchange between misaligned DNA sequences. The model predicts that misalignments in one chromosomal region can influence the nature of mutations in another region. Thus, as often has been observed, the mutability of a base pair will be determined by its location in a DNA sequence. Furthermore, the intrachromosomal recombination of DNA sequences may account for complex genetic modifications that occur as new pathways evolve. The model provides an interpretation of an apparent paradox, the rapid creation of new metabolic traits by bacterial genomes that are remarkably resistant to genetic drift.

p-Chloromercuribenzoate specifically modifies thiols associated with the active sites of beta-ketoadipate enol-lactone hydrolase and succinyl CoA: beta-ketoadipate CoA transferase.

beta-Ketoadipate enol-lactone hydrolase (EC 3.1.1.24) and succinyl CoA: beta-ketoadipate transferase (EC 2.8.3.6) catalyze consecutive metabolic reactions in bacteria. The enzymes appear to be members of different families of related proteins. Enzymes within the enol-lactone hydrolase family appear to have diverged so extensively that common ancestry sometimes is not directly evident from comparison of NH2-terminal amino acid sequences of the proteins. Amino acid sequences at or near the active sites of the enzymes are likely to have been conserved, and hence a chemical proble that reacted specifically near the active sites of the enzymes might identify regions of amino acid sequence in which evolutionary affinities among widely divergent proteins could be identified. p-Chloromercuribenzoate appears to be such a probe because enol-lactone hydrolases and CoA transferases from Acinetobacter calcoaceticus and Pseudomonas putida were completely inhibited by stoichiometric quantities of the compound which appears to modify selectively cysteinyl side chains at or near the active sites of the enzymes. Stoichiometric inhibition of P. putida enol-lactone hydrolase was observed in the presence of excess dithiothreitol; therefore the reactive cysteinyl residue in this enzyme appears to be nucleophilic. The hydrolase is inhibited by beta-ketoadipate, but the compound must be supplied at 10 mM concentrations in order to achieve 50% inhibition, so the product inhibition is unlikely to be significant under physiological conditions.

Two distinctive O-methyltransferases catalyzing penultimate and terminal reactions of macrolide antibiotic (tylosin) biosynthesis. Substrate specificity, enzyme inhibition, and kinetic mechanism.

S-Adenosyl-L-methionine:demethylmacrocin O-methyltransferase catalyzes the conversion of demethylmacrocin to macrocin as the penultimate step of tylosin biosynthesis in Streptomyces fradiae. The O-methyltransferase was purified to electrophoretic homogeneity by a conventional chromatographic procedure. The purified enzyme appears to be trimeric with a molecular weight of 122,000-126,000 and a subunit size of 42,000. Its isoelectric point was 6.0. The enzyme required Mg2+ for maximal activity and was catalytically optimal at pH 7.8-8.5 and 42 degrees C. The O-methyltransferase catalyzed conversion of demethylmacrocin to macrocin at a stoichiometric ratio of 1:1. The O-methyltransferase also mediated conversion of demethyllactenocin----lactenocin. The corresponding Vmax/Km ratios for the two analogous conversions varied only slightly. Both enzymic conversions were susceptible to an extensive and identical range of metabolic inhibitions. Steady-state kinetic studies for initial velocity, substrate analogue, and product inhibitions are consistent with Ordered Bi Bi as the reaction mechanism of demethylmacrocin O-methyltransferase. Except for an identical kinetic mechanism, demethylmacrocin O-methyltransferase can be readily differentiated from macrocin O-methyltransferase by its physical and catalytic properties as well as metabolic inhibitions.

Purification and properties of NADPH-dependent tylosin reductase from Streptomyces fradiae.

A reductase of Streptomyces fradiae was speculated to catalyze reduction of tylosin to relomycin, an industrially undesirable product. The activity of tylosin reductase was closely related to bacterial growth, suggesting involvement of the enzyme in a primary metabolism. The reductase activity was improved significantly in vivo and in vitro. The enzyme was also partially stabilized in vitro. Using a simple five-step chromatographic procedure, the reductase was purified 480-fold to apparent homogeneity. The purified reductase had a molecular mass of 270 kDa and consisted of two different subunits of 26 and 7 kDa at 1:1 ratio. The enzyme exhibited an absorption maximum at 405 nm and was inhibited by exogenous FAD or FMN, indicating a flavin as its prosthetic group. Tylosin reductase was optimally active at pH 7.0-7.2 and 40 degrees C with NADPH as a preferred electron donor. The Km of the enzyme for tylosin was 1.4 mM and that for NADPH was 0.15 mM. The Vmax for the enzymatic reaction was 917 mumol of tylosin formed/min/mg protein. The enzymatic conversion of tylosin to relomycin was coupled to that of NADPH to NADP+ at a stoichiometric ratio of 1:1. Tylosin reductase showed a broad substrate specificity toward all macrolide aldehydes (as normal and shunt metabolites of tylosin biosynthesis) tested. Thus, the enzyme may have a physiological role of macrolide detoxification for the bacterium.

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.

Overlapping evolutionary affinities revealed by comparison of amino acid compositions.

Comparison of the amino acid compositions of purified proteins indicates the presence of overlapping evolutionary affinities among enzymes of the beta-ketoadipate pathway. Isofunctional enzymes from different bacterial genera share a common evolutionary origin. Moreover, enzymes that mediate isofunctional or chemically analogous reactions within an organism appear to be evolutionarily homologous. Most remarkably, closely similar amino acid compositions are found in enzymes that mediate the following consecutive metabolic steps: lactonization, decarboxylation, hydrolysis, and transfer of a thioester bond.

Evolutionary divergence of co-selected beta-ketoadipate enol-lactone hydrolases in Acinetobacter calcoaceticus.

Muconolactone isomerase (EC 5.3.3.4) and beta-ketoadipate enol-lactone hydrolase (EC 3.1.1.24) mediate consecutive catabolic steps in bacteria. Separately inducible beta-ketoadipate enol-lactone hydrolases I and II are formed in representatives of Acinetobacter calcoaceticus. When subjected to DEAE-cellulose chromatography, Acinetobacter enol-lactone hydrolase I displays heterogeneous behavior which, in whole or in part, appears to be due to modifications of sulfhydryl groups in the protein; the enzyme is unusual in that its NH2-terminal amino acid is cysteine. Comparison of the NH2-terminal amino acid sequence of Acinetobacter enollactone hydrolase I, reported here, with the corresponding amino acid sequences of Acinetobacter enollactone hydrolase II and Pseudomonas enol-lactone hydrolase indicates that all three proteins have diverged widely from a common evolutionary origin. Sequence comparisons suggest that divergence of the Acinetobacter enol-lactone hydrolase structural genes was achieved by substitution with DNA derived from an ancestral muconolactone isomerase structural gene.

Homologies in the NH2-terminal amino acid sequences of gamma-carboxymuconolactone decarboxylases and muconolactone isomerases.

gamma-Carboxymuconolactone decarobxylase (EC 4.1.1.44) and muconolactone isomerase (EC 5.3.3.4) mediate chemically analogous reactions in bacteria. The enzymes are inducible, and different metabolites trigger the respective syntheses of the decarboxylases in Acinetobacter calcoaceticus and Pseudomonas putida. The decarobxylases share similar oligomeric structures in which identical subunits of about 13,300 daltons appear to be self-associated into hexamers. Identical residues are found in 18 of the first 36 positions of the enzymes' NH2-terminal amino acid sequences. Thus, genetic rearrangements appear to have placed homologous structural genes for the decarboxylases under different transcriptional control in the two bacterial species. The NH2-terminal amino acid sequences of the decarboxylases and muconolactone isomerases are similar, suggesting that a common ancestral protein gave rise to the enzymes with different (albeit analogous) activities. In addition, the NH2-terminal amino acid sequences of the decarboxylases appear to have been conserved at a second region within the primary structure of the muconolactone isomerases. As has been observed with the two enol-lactone hydrolases (EC 3.1.3.24) of Acinetobacter, the structural genes for the decarboxylases and the isomerases appear to have diverged widely as they were co-selected within a single cell line, In part the divergence appears to have been achieved by mutations in which fragments of DNA within structural genes are replaced with fragments of DNA derived from a co-evolving sequence.

High-performance liquid chromatographic assay for S-adenosyl-L-methionine: macrocin O-methyltransferase.

A high-performance liquid chromatographic (HPLC) procedure was developed to assay S-adenosyl-L-methionine: macrocin O-methyltransferase. This enzyme catalyzes the rate-limiting terminal reaction of tylosin biosynthesis in Streptomyces fradiae. HPLC analysis was improved by resin treatment of cell-free extracts to remove endogenous tylosin and related compounds. Relomycin was selected as an internal standard and the enzymatic reaction conditions were optimized. The reaction mixture was extracted with ethyl acetate to recover the substrate, product and the internal standard. Efficient separation of the macrolide antibiotics was provided by ion-pair reversed-phase HPLC. An average relomycin recovery was 90%. The O-methyltransferase activity could be routinely and reproducibly determined by monitoring tylosin formation at 285 nm.

Purification, characterization, and kinetic mechanism of S-adenosyl-L-methionine:macrocin O-methyltransferase from Streptomyces fradiae.

S-Adenosyl-L-methionine:macrocin O-methyltransferase catalyzes conversion of macrocin to tylosin, the terminal and main rate-limiting step of tylosin biosynthesis in Streptomyces fradiae. The O-methyltransferase was stabilized in vitro and purified to electrophoretic homogeneity. The purified enzyme had a molecular weight of 65,000 and consisted of two identical subunits of 32,000 with an isoelectric point of 4.5. The enzyme required Mg2+, Mn2+, or Co2+ for maximal activity and was catalytically optimal at pH 7.5-8.0 and 31 degrees C. The O-methyltransferase catalyzed the conversion of macrocin to tylosin at a stoichiometric ratio of 1:1. The enzyme also mediated conversion of lactenocin----desmycosin. The corresponding Vmax/Km ratios for the two analogous conversions were similar, and both enzymic conversions were susceptible to extensive competitive and noncompetitive inhibitions by macrolide metabolites. Steady-state kinetic studies for initial velocity, substrate analogue, and product inhibitions have allowed formulation of Ordered Bi Bi as the reaction mechanism for macrocin O-methyltransferase.

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

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.