Prokaryotic Enzymes Research Articles

An assay for bacterial and eukaryotic chondroitinases using a chondroitin sulfate-binding protein

A simple, rapid, and reproducible microtiter-based chondroitinase (CSase) assay is reported here, based on the competition of chondroitin sulfate (CS) with immobilized hyaluronan (HA) for the binding of TSG-6 protein, the product of TNF-inducible gene 6. Although the catabolic reaction of bacterial and other prokaryotic CSase enzymes, often referred to as the chondroitin lyases, can be followed by tracking the generation of unsaturated bonds by the spectrophotometrical determination of the absorbance at 232 nm, no rapid, sensitive, and simple assay has been devised to date for measuring the activity of the vertebrate enzymes that cleave their substrate exclusively by hydrolysis. We provide data demonstrating that the CSase assay described here is suitable for the determination of the activities of both classes of enzymes. For the bacterial enzyme CSase ABC, both the determination of the absorbance at 232 nm and the assay based on TSG-6 binding are suitable using the same range of enzyme activities. However, for testicular hyaluronidase, considerably higher enzyme activities were needed to cleave CS than to cleave HA. Using the HA-binding domain of aggrecan for a comparison, we determined that the interaction between TSG-6 and chondroitin sulfate is uniquely suited for this CSase assay.

Purification and characterization of recombinant Caulobacter crescentus Cu,Zn superoxide dismutase

Recombinant Cu,Zn Superoxide Dismutase from Caulobacter crescentus has been expressed in Escherichia coli and characterized. The corresponding recombinant protein has a molecular weight typical of a homodimeric Cu,ZnSODs and an activity comparable to that of other prokaryotic enzymes. The copper active site is characterized by a peculiar axial geometry as evidenced by its electron paramagnetic resonance spectrum, moreover, the copper atom displays a low accessibility toward external chelating agents indicating a lower solvent accessibility when compared to other prokaryotic enzymes. Investigation of the enzyme thermal stability through differential scanning calorimetry indicates the occurrence of two transitions at low and higher temperature that are found to be due to the apo and holo protein, respectively, confirming that the metals have a crucial role in the stabilization of this class of enzymes.

Epoxide Formation on the Aromatic B Ring of Flavanone by Biphenyl Dioxygenase of Pseudomonas pseudoalcaligenes KF707

Prokaryotic dioxygenase is known to catalyze aromatic compounds into their corresponding cis-dihydrodiols without the formation of an epoxide intermediate. Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 showed novel monooxygenase activity by converting 2(R)- and 2(S)-flavanone to their corresponding epoxides (2-(7-oxabicyclo[4.1.0]hepta-2,4-dien-2-yl)-2, 3-dihydro-4H-chromen-4-one), whereby the epoxide bond was formed between C2' and C3' on the B ring of the flavanone. The enzyme also converted 6-hydroxyflavanone and 7-hydroxyflavanone, which do not contain a hydroxyl group on the B-ring, to their corresponding epoxides. In a previous report (S.-Y. Kim, J. Jung, Y. Lim, J.-H. Ahn, S.-I. Kim, and H.-G. Hur, Antonie Leeuwenhoek 84:261-268, 2003), however, we found that the same enzyme showed dioxygenase activity toward flavone, resulting in the production of flavone cis-2',3'-dihydrodiol. Extensive structural identification of the metabolites of flavanone by using high-pressure liquid chromatography, liquid chromatography/mass spectrometry, and nuclear magnetic resonance confirmed the presence of an epoxide functional group on the metabolites. Epoxide formation as the initial activation step of aromatic compounds by oxygenases has been reported to occur only by eukaryotic monooxygenases. To the best of our knowledge, biphenyl dioxygenase from P. pseudoalcaligenes KF707 is the first prokaryotic enzyme detected that can produce an epoxide derivative on the aromatic ring structure of flavanone.

Investigation of the catalytic triad of arylamine N-acetyltransferases: essential residues required for acetyl transfer to arylamines.

The NATs (arylamine N-acetyltransferases) are a well documented family of enzymes found in both prokaryotes and eukaryotes. NATs are responsible for the acetylation of a range of arylamine, arylhydrazine and hydrazine compounds. We present here an investigation into the catalytic triad of residues (Cys-His-Asp) and other structural features of NATs using a variety of methods, including site-directed mutagenesis, X-ray crystallography and bioinformatics analysis, in order to investigate whether each of the residues of the catalytic triad is essential for catalytic activity. The catalytic triad of residues, Cys-His-Asp, is a well defined motif present in several families of enzymes. We mutated each of the catalytic residues in turn to investigate the role they play in catalysis. We also mutated a key residue, Gly126, implicated in acetyl-CoA binding, to examine the effects on acetylation activity. In addition, we have solved the structure of a C70Q mutant of Mycobacterium smegmatis NAT to a resolution of 1.45 A (where 1 A=0.1 nm). This structure confirms that the mutated protein is correctly folded, and provides a structural model for an acetylated NAT intermediate. Our bioinformatics investigation analysed the extent of sequence conservation between all eukaryotic and prokaryotic NAT enzymes for which sequence data are available. This revealed several new sequences, not yet reported, of NAT paralogues. Together, these studies have provided insight into the fundamental core of NAT enzymes, and the regions where sequence differences account for the functional diversity of this family. We have confirmed that each of the three residues of the triad is essential for acetylation activity.

Is a third proton-conducting pathway operative in bacterial cytochrome c oxidase?

Despite the existence of several three-dimensional structures of cytochrome c oxidases, a detailed understanding of pathways involved in proton movements through the complex remains largely elusive. Next to the two well-established pathways (termed D and K), an additional proton-conducting network ('H-channel') has been proposed for the beef heart enzyme. Yet, our recent mutational studies on corresponding residues of the Paracoccus denitrificans cytochrome c oxidase provide no clues that such a pathway operates in the prokaryotic enzyme.

Calorimetric and Crystallographic Analysis of the Oligomeric Structure of Escherichia coli GMP Kinase

Guanosine monophosphate kinases (GMPKs), which catalyze the phosphorylation of GMP and dGMP to their diphosphate form, have been characterized as monomeric enzymes in eukaryotes and prokaryotes. Here, we report that GMPK from Escherichia coli (ecGMPK) assembles in solution and in the crystal as several different oligomers. Thermodynamic analysis of ecGMPK using differential scanning calorimetry shows that the enzyme is in equilibrium between a dimer and higher order oligomers, whose relative amounts depend on protein concentration, ionic strength, and the presence of ATP. Crystallographic structures of ecGMPK in the apo, GMP and GDP-bound forms were solved at 3.2A, 2.9A and 2.4A resolution, respectively. ecGMPK forms a hexamer with D3 symmetry in all crystal forms, in which the two nucleotide-binding domains are able to undergo closure comparable to that of monomeric GMPKs. The 2-fold and 3-fold interfaces involve a 20-residue C-terminal extension and a sequence signature, respectively, that are missing from monomeric eukaryotic GMPKs, explaining why ecGMPK forms oligomers. These signatures are found in GMPKs from proteobacteria, some of which are human pathogens. GMPKs from these bacteria are thus likely to form the same quaternary structures. The shift of the thermodynamic equilibrium towards the dimer at low ecGMPK concentration together with the observation that inter-subunit interactions partially occlude the ATP-binding site in the hexameric structure suggest that the dimer may be the active species at physiological enzyme concentration.

Acetyl-coenzyme A carboxylase: crucial metabolic enzyme and attractive target for drug discovery

Acetyl-coenzyme A carboxylases (ACCs) have crucial roles in fatty acid metabolism in most living organisms. Mice deficient in ACC2 have continuous fatty acid oxidation and reduced body fat and body weight, validating this enzyme as a target for drug development against obesity, diabetes and other symptoms of the metabolic syndrome. ACC is a biotin-dependent enzyme and catalyzes the carboxylation of acetyl-CoA to produce malonyl-CoA through its two catalytic activities, biotin carboxylase (BC) and carboxyltransferase (CT). ACC is a multi-subunit enzyme in most prokaryotes, whereas it is a large, multi-domain enzyme in most eukaryotes. The activity of the enzyme can be controlled at the transcriptional level as well as by small molecule modulators and covalent modification. This review will summarize the structural information that is now available for both the BC and CT enzymes, as well as the molecular mechanism of action of potent ACC inhibitors. The current intense research on these enzymes could lead to the development of novel therapies against metabolic syndrome and other diseases.

Critical sources of error in colorimetric assay for UDP- N-acetylglucosamine pyrophosphorylase

UDP-N-acetylglucosamine pyrophosphorylase (UAP)1 is a key enzyme in prokaryotes and eukaryotes leading to synthesis of a ubiquitous metabolite, UDP-N-acetylglucosamine (UDP-N-GlcNAc), for polymerization. This enzyme catalyzes the reaction UTP+ N-acetylglucosamine-1-phosphate (GlcNAc-1-P)!UDP-GlcNAc+ pyrophosphate (PPi), and its activity was usually determined by a radioactive assay in which product from a radioactive substrate was identiWed by chromatography and measured by a scintillation counter [1–4]. However, this assay has major concerns about safety, complicated handling, and availability of radioactive substrates. Mio et al. [5] mentioned another strategy in which a coupling enzyme, pyrophosphatase, is included in the anabolic reaction to convert the pyrophosphate into inorganic phosphate that can be readily quantiWed by a malachite green colorimetric assay. Nevertheless, the precautions of this assay have never been discussed, and ignorance of proper controls was found to result in signiWcant artifacts. The current report describes the basic principle, reaction condition, and critical sources of error of this colorimetric assay to allow accurate and reliable measurement of UAP activity. The principle of the malachite green colorimetric assay is based on the reaction between phosphate and molybdate to form a phosphomolybdate complex that binds to the dye and induces the color changes [6]. It is

The prokaryotic enzyme DsbB may share key structural features with eukaryotic disulfide bond forming oxidoreductases

Three different classes of thiol-oxidoreductases that facilitate the formation of protein disulfide bonds have been identified. They are the Ero1 and SOX/ALR family members in eukaryotic cells, and the DsbB family members in prokaryotic cells. These enzymes transfer oxidizing potential to the proteins PDI or DsbA, which are responsible for directly introducing disulfide bonds into substrate proteins during oxidative protein folding in eukaryotes and prokaryotes, respectively. A comparison of the recent X-ray crystal structure of Ero1 with the previously solved structure of the SOX/ALR family member Erv2 reveals that, despite a lack of primary sequence homology between Ero1 and Erv2, the core catalytic domains of these two proteins share a remarkable structural similarity. Our search of the DsbB protein sequence for features found in the Ero1 and Erv2 structures leads us to propose that, in a fascinating example of structural convergence, the catalytic core of this integral membrane protein may resemble the soluble catalytic domain of Ero1 and Erv2. Our analysis of DsbB also identified two new groups of DsbB proteins that, based on sequence homology, may also possess a catalytic core similar in structure to the catalytic domains of Ero1 and Erv2.

DNA-[Adenine] Methylation in Lower Eukaryotes

DNA methylation in lower eukaryotes, in contrast to vertebrates, can involve modification of adenine to N6-methyladenine (m6A). While DNA-[cytosine] methylation in higher eukaryotes has been implicated in many important cellular processes, the function(s) of DNA-[adenine] methylation in lower eukaryotes remains unknown. I have chosen to study the ciliate Tetrahymena thermophila as a model system, since this organism is known to contain m6A, but not m5C, in its macronuclear DNA. A BLAST analysis revealed an open reading frame (ORF) that appears to encode for the Tetrahymena DNA-[adenine] methyltransferase (MTase), based on the presence of motifs characteristic of the enzymes in prokaryotes. Possible biological roles for DNA-[adenine] methylation in Tetrahymena are discussed. Experiments to test these hypotheses have begun with the cloning of the gene. Orthologous ORFs are also present in three species of the malarial parasite Plasmodium. They are compared to one another and to the putative Tetrahymena DNA-[adenine] MTase. The gene from the human parasite P. falciparum has been cloned.

Nucleotide-dependent Formation of Catalytically Competent Dimers from Engineered Monomeric Ribonucleotide Reductase Protein R1

Each catalytic turnover by aerobic ribonucleotide reductase requires the assembly of the two proteins, R1 (alpha(2)) and R2 (beta(2)), to produce deoxyribonucleotides for DNA synthesis. The R2 protein forms a tight dimer, whereas the strength of the R1 dimer differs between organisms, being monomeric in mouse R1 and dimeric in Escherichia coli. We have used the known E. coli R1 structure as a framework for design of eight different mutations that affect the helices and proximal loops that comprise the dimer interaction area. Mutations in loop residues did not affect dimerization, whereas mutations in the helices had very drastic effects on the interaction resulting in monomeric proteins with very low or no activity. The monomeric N238A protein formed an interesting exception, because it unexpectedly was able to reduce ribonucleotides with a comparatively high capacity. Gel filtration studies revealed that N238A was able to dimerize when bound by both substrate and effector, a result in accordance with the monomeric R1 protein from mouse. The effects of the N238A mutation, fit well with the notion that E. coli protein R1 has a comparatively small dimer interaction surface in relation to its size, and the results illustrate the stabilization effects of substrates and effectors in the dimerization process. The identification of key residues in the dimerization process and the fact that there is little sequence identity between the interaction areas of the mammalian and the prokaryotic enzymes may be of importance in drug design, similar to the strategy used in treatment of HSV infection.

Improvement of the thermostability and catalytic activity of a mesophilic family 11 xylanase by N-terminus replacement

To improve the thermostability and catalytic activity of Aspergillus niger xylanase A (AnxA), its N-terminus was substituted with the corresponding region of Thermomonospora fusca xylanase A (TfxA). The constructed hybrid xylanase, named ATx, was overexpressed in Pichia pastoris and secreted into the medium. After 96-h 0.25% methanol induction, the activity of the ATx in the culture supernatant reached its peak, 633 U/mg, which was 3.6 and 5.4 times as high as those of recombinant AnxA (reAnxA) and recombinant TfxA (reTfxA), respectively. Studies on enzymatic properties showed that the temperature and pH optimum of the ATx were 60 °C and 5.0, respectively. The ATx was more thermostable, when it was treated at 70 °C, pH 5.0, for 2 min, the residual activity was 72% which was higher than that of reAnxA and similar to that of reTfxA. The ATx was very stable over a broader pH range (3.0–10.0) and much less affected by acid/base conditions. After incubation at pH 3.0–10.0, 25 °C for 1 h, all the residual activities of the ATx were over 80%. These results revealed that the thermostability and catalytic activity of the AnxA were enhanced. The N-terminus of TfxA contributed to the observed thermostability of itself and the ATx, and to the high activity of the ATx. Replacement of N-terminus between mesophilic eukaryotic and thermostable prokaryotic enzymes may be a useful method for constructing the new and improved versions of biologically active enzymes.

Carbonic anhydrase in relation to higher plants

The review incorporates recent information on carbonic anhydrase (CA, EC: 4.2.1.1) pertaining to types, homology, regulation, purification, in vitro stability, and biological functions with special reference to higher plants. CA, a ubiquitous enzyme in prokaryotes and higher organisms represented by four distinct families, is involved in diverse biological processes, including pH regulation, CO2 transfer, ion exchange, respiration, and photosynthetic CO2 fixation. CA from higher plants traces its origin with prokaryotes and exhibits compartmentalization among their organs, tissues, and cellular organelles commensurate with specific functions. In leaves, CA represents 1−20 % of total soluble protein and abundance next only to ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) in chloroplast, facilitating CO2 supply to phosphoenol pyruvate carboxylase in C4 and CAM plants and RuBPCO in C3 plants. It confers special significance to CA as an efficient biochemical marker for carbon sequestration and environmental amelioration in the current global warming scenario linked with elevated CO2 concentrations.

The alternative oxidase from Euglena gracilis

The alternative oxidase (AOX) is an ubiquinol oxidase of the mitochondrial respiratory chain. AOX is an additional terminal oxidase; the electron flux to AOX branches from the classical electron‐transport chain at the level of the ubiquinone pool leading to the direct reduction of oxygen to water in a single four‐electron transfer step. O2 reduction catalyzed by AOX is not coupled to proton pumping and hence not ATP‐producing. The energy is released as heat. AOX is specifically inhibited by salicylhydroxamic acid (SHAM), but is cyanide resistant. AOX was discovered in plant thermogenic tissues and all plant species tested to date can express the alternative pathway. It was long thought that AOX is specific to higher plants, but it is now turning up in many non‐photosynthetic eukaryotes. For example the bloodstream form of the protist Trypanosoma brucei brucei that lacks functional cytochromes, limits most of its energy metabolism to glycolysis while passing the reducing equivalents produced to water via a plant‐like alternative oxidase. It has also been reported that oxidative phosphorylation in the protist Euglena gracilis is supported by an alternative respiratory pathway in which an AOX is involved. We have cloned the mitochondrial AOX from Euglena and have investigated the evolutionary history of this enzyme in prokaryotes and eukaryotes.

Bismuth–dithiol inhibition of the Escherichia coli rho transcription termination factor

Bismuth–dithiol mixtures are proven antimicrobial agents with unknown mechanism(s) of action. We show that select bismuth–dithiol solutions inhibit the Escherichia coli rho transcription termination factor. Rho is an essential enzyme in most Gram-negative prokaryotes and without rho function the cells are not viable. Bismuth complexes with 2,3-dimercapto-1-propanol (BiBAL) (3:1 solutions) functioned as a noncompetitive inhibitor with respect to ATP in the rho poly(C)-dependent ATPase assay ( I 50 = 60 μM) and as a competitive inhibitor with respect to ribo(C) 10 in the poly(dC)-ribo(C) 10-dependent ATPase assay. The minimum inhibitory concentration (MIC) of bacterial growth for BiBAL (3:1) in the liquid culture assay using E. coli W3350 was 16 μM. Using the tnaA/ lacZ fusion reporter assay we showed that sublethal amounts (3 μM) of BiBAL (3:1 solution) led to a small increase (37%) in in vivo β-glactosidase activity in E. coli SVS1144, which corresponds to antitermination of the tna operon as a result of rho inhibition. We concluded that BiBAL was a potent in vitro rho inhibitor but its effect on in vivo rho processes was modest indicating that other mechanisms contributed to the antibacterial activity of BiBAL. Our study suggests that structural changes in the dithiol unit that provide greater bismuth binding may improve rho specificity, a macromolecular target not previously recognized for bismuth therapy.

A Novel Enzyme Complex of Orotate Phosphoribosyltransferase and Orotidine 5‘-Monophosphate Decarboxylase in Human Malaria Parasite Plasmodium falciparum: Physical Association, Kinetics, and Inhibition Characterization,

Human malaria parasite, Plasmodium falciparum, can only synthesize pyrimidine nucleotides using the de novo pathway, whereas mammalian cells obtain pyrimidine nucleotides from both the de novo and salvage pathways. The parasite's orotate phosphoribosyltransferase (PfOPRT) and orotidine 5'-monophosphate decarboxylase (PfOMPDC) of the de novo pyrimidine pathway are attractive targets for antimalarial drug development. Previously, we have reported that the two enzymes in P. falciparum exist as a multienzyme complex containing two subunits each of 33-kDa PfOPRT and 38-kDa PfOMPDC. In this report, the gene encoding PfOPRT has been cloned and expressed in Escherichia coli. An open reading frame of PfOMPDC gene was identified in the malaria genome database, and PfOMPDC was cloned from P. falciparum cDNA, functionally expressed in E. coli, purified, and characterized. The protein sequence has <20% identity with human OMPDC and four microbial OMPDC for which crystal structures are known. Recombinant PfOMPDC was catalytically active in a dimeric form. Both recombinant PfOPRT and PfOMPDC monofunctional enzymes were kinetically different from the native multienzyme complex purified from P. falciparum. Oligomerization of PfOPRT and PfOMPDC cross-linked by dimethyl suberimidate indicated that they were tightly associated as the heterotetrameric 140-kDa complex, (PfOPRT)2(PfOMPDC)2. Kinetic analysis of the PfOPRT-PfOMPDC associated complex was similar to that of the native P. falciparum enzymes and was different from that of the bifunctional human enzymes. Interestingly, a nanomolar inhibitor of the yeast OMPDC, 6-thiocarboxamido-uridine 5'-monophosphate, was about 5 orders of magnitude less effective on the PfOMPDC than on the yeast enzyme. Our results support that the malaria parasite has unique structural and functional properties, sharing characteristics of the monofunctional pyrimidine-metabolizing enzymes in prokaryotes and bifunctional complexes in eukaryotes.

Expression, purification, characterization and structure of Pseudomonas aeruginosa arylamine N-acetyltransferase.

The gene for NAT (arylamine N-acetyltransferase) from Pseudomonas aeruginosa (panat) has been cloned from genomic DNA, and the gene product (PANAT) expressed as an N-terminal histidine-tagged protein in Escherichia coli and purified via nickel ion affinity chromatography. The specific activities of PANAT against a broad range of substrates have been investigated and compared with those of other prokaryotic NAT enzymes. For most arylamine substrates identified, PANAT exhibits in vitro specific activities typically one order of magnitude greater than those of recombinant NAT enzymes from Mycobacterium smegmatis or Salmonella typhimurium. Among the substrates of PANAT so far identified are the anti-tubercular drug isoniazid, 5-aminosalicylate (a drug used in the treatment of inflammatory bowel disease), as well as important environmental pollutants such as 3,4-dichloroaniline and 2-aminofluorene. As well as acetylating common NAT substrates, PANAT is unique among the prokaryotic NATs so far studied in acetylating the folate precursor 4-aminobenzoic acid and the folate catabolite 4-aminobenzoylglutamate. The recombinant protein has been expressed in sufficient quantity to allow protein crystallization, and we have subsequently determined the 1.95 A structure of PANAT by X-ray crystallography.

Electron Transfer Ability from NADH to Menaquinone and from NADPH to Oxygen of Type II NADH Dehydrogenase ofCorynebacterium glutamicum

Type II NADH dehydrogenase of Corynebacterium glutamicum (NDH-2) was purified from an ndh overexpressing strain. Purification conferred 6-fold higher specific activity of NADH:ubiquinone-1 oxidoreductase with a 3.5-fold higher recovery than that previously reported (K. Matsushita et al., 2000). UV-visible and fluorescence analyses of the purified enzyme showed that NDH-2 of C. glutamicum contained non-covalently bound FAD but not covalently bound FMN. This enzyme had an ability to catalyze electron transfer from NADH and NADPH to oxygen as well as various artificial quinone analogs at neutral and acidic pHs respectively. The reduction of native quinone of C. glutamicum, menaquinone-2, with this enzyme was observed only with NADH, whereas electron transfer to oxygen was observed more intensively with NADPH. This study provides evidence that C. glutamicum NDH-2 is a source of the reactive oxygen species, superoxide and hydrogen peroxide, concomitant with NADH and NADPH oxidation, but especially with NADPH oxidation. Together with this unique character of NADPH oxidation, phylogenetic analysis of NDH-2 from various organisms suggests that NDH-2 of C. glutamicum is more closely related to yeast or fungal enzymes than to other prokaryotic enzymes.

ProLysED

Bacterial proteases are an important group of enzymes that have very diverse biochemical and cellular functions. Proteases from prokaryotic sources also have a wide range of uses, either in medicine as pathogenic factors or in industry and therapeutics. ProLysED (Prokaryotic Lysis Enzymes Database), our meta-server integrated database of bacterial proteases, is a useful, albeit very niche, resource. The features include protease classification browsing and searching, organism-specific protease browsing, molecular information and visualisation of protease structures from the Protein Data Bank (PDB) as well as predicted protease structures. ProLysED is integrated into the ProLysES (Prokaryotic Lysis Enzymes Site) website at http://genome.ukm.my/prolyses/. Access to the ProLysED database is free for academic users upon registration.

A Mutation in Escherichia coli DNA Gyrase Conferring Quinolone Resistance Results in Sensitivity to Drugs Targeting Eukaryotic Topoisomerase II

Fluoroquinolones are broad-spectrum antimicrobial agents that target type II topoisomerases. Many fluoroquinolones are highly specific for bacterial type II topoisomerases and act against both DNA gyrase and topoisomerase IV. In Escherichia coli, mutations causing quinolone resistance are often found in the gene that encodes the A subunit of DNA gyrase. One common site for resistance-conferring mutations alters Ser83, and mutations to Leu or Trp result in high levels of resistance to fluoroquinolones. In the present study we demonstrate that the mutation of Ser83 to Trp in DNA gyrase (Gyr(S83W)) also results in sensitivity to agents that are potent inhibitors of eukaryotic topoisomerase II but that are normally inactive against prokaryotic enzymes. Epipodophyllotoxins, such as etoposide, teniposide and amino-azatoxin, inhibited the DNA supercoiling activity of Gyr(S83W), and the enzyme caused elevated levels of DNA cleavage in the presence of these agents. The DNA sequence preference for Gyr(S83W)-induced cleavage sites in the presence of etoposide was similar to that seen with eukaryotic type II topoisomerases. Introduction of the Gyr(S83W) mutation in E. coli strain RFM443-242 by site-directed mutagenesis sensitized it to epipodophyllotoxins and amino-azatoxin. Our results demonstrate that sensitivity to agents that target topoisomerase II is conserved between prokaryotic and eukaryotic enzymes, suggesting that drug interaction domains are also well conserved and likely occur in domains important for the biochemical activities of the enzymes.