Valerie Kramlinger - Academia.edu (original) (raw)
Papers by Valerie Kramlinger
Drug metabolism letters
Timolol is clinically administered topically (ocular) to reduce intraocular pressure and treat op... more Timolol is clinically administered topically (ocular) to reduce intraocular pressure and treat open-angle glaucoma. Ocular administration of timolol in low doses (0.5% w/v in the form of eye drops) has led to challenges for in vivo metabolite identification. An understanding of drug metabolism in the eye is important for clinical ocular therapeutics and potential drug candidates. We aimed to investigate the metabolism of timolol in rat ocular and liver S9 fractions, as well as rat ocular tissue and plasma following a 0.5% topical (ocular) dose of timolol. We explored the potential in vitro metabolic bioactivation in the eye/liver by conducting trapping studies for putative aldehyde and iminium ion intermediates that may arise from the morpholine functionality. Oxidative metabolism of timolol to its major metabolite (M4) in ocular S9 and recombinant rat cytochrome P450 (CYP) isoforms supports the possible role of rat ocular CYP2D2, 2D4, and/or 2D18. Observation of N-acetyl-timolol (M...
Journal of Pharmaceutical Sciences
Nicotine, the primary addictive compound in cigarettes, is metabolized in humans by cytochrome P4... more Nicotine, the primary addictive compound in cigarettes, is metabolized in humans by cytochrome P450 2A enzymes. The hepatic enzyme responsible for the metabolism of nicotine in smokers is P450 2A6. P450 2A13, which shares 94% primary sequence homology with P450 2A6, also catalyzes the metabolism of nicotine and is present in the lung. Loss of P450 2A activity is correlated with modified smoking behavior and addiction. Inhibition of these enzymes decreases nicotine metabolism and may be of benefit in smoking cessation. This thesis investigates tobacco-related molecules that may impact P450 2A function and is presented in three parts. In the first, the potential inhibitory potency of (-)-menthol, (R)-(+)-menthofuran, and -nicotyrine of both P450s 2A6 and 2A13 are investigated. All three compounds inhibit P450 2A6 and 2A13 activity. In addition, menthofuran and -nicotyrine mediate mechanism-based inactivation of P450 2A6 but not 2A13. Second, the P450 2A6 and P450 2A13-mediated metabolism of -nicotyrine is studied and three metabolites are identified. -nicotyrine is readily turned over by both P450 2A6 and P450 2A13 as indicated by the calculated K m (4.4µM and 5.0µM, respectively) and V max (21 and 37 pmol product/min/pmol, respectively) values. Also in the second section, -nicotyrine is shown to be a metabolite of P450 2A6-mediated nicotine metabolism. In the last section, attempts to identify a covalent adduct that would result from menthofuran or -nicotyrine-mediated inactivation are presented, but these are largely unsuccessful.
FEBS Letters, 2016
In humans, a considerable fraction of the retinoid pool in skin is derived from vitamin A2 (all-t... more In humans, a considerable fraction of the retinoid pool in skin is derived from vitamin A2 (all-trans 3,4-dehydroretinal). Vitamin A2 may be locally generated by keratinocytes, which can convert vitamin A1 (all-trans retinol) into vitamin A2 in cell culture. We report that human cytochrome P450 (hP450) 27C1, a previously 'orphan' enzyme, can catalyze this reaction. Purified recombinant hP450 27C1 bound and desaturated all-trans retinol, retinal, and retinoic acid, as well as 11-cis retinal. Although the physiological role of 3,4-dehydroretinoids in humans is unclear, we have identified hP450 27C1 as an enzyme capable of efficiently mediating their formation. This article is protected by copyright. All rights reserved.
Current Biology, 2015
Some vertebrate species have evolved means of extending their visual sensitivity beyond the range... more Some vertebrate species have evolved means of extending their visual sensitivity beyond the range of human vision. One mechanism of enhancing sensitivity to long-wavelength light is to replace the 11-cis retinal chromophore in photopigments with 11-cis 3,4-didehydroretinal. Despite over a century of research on this topic, the enzymatic basis of this perceptual switch remains unknown. Here, we show that a cytochrome P450 family member, Cyp27c1, mediates this switch by converting vitamin A1 (the precursor of 11-cis retinal) into vitamin A2 (the precursor of 11-cis 3,4didehydroretinal). Knockout of cyp27c1 in zebrafish abrogates production of vitamin A2, eliminating the animal's ability to red-shift its photoreceptor spectral sensitivity and reducing its ability to see and respond to near-infrared light. Thus, the expression of a single enzyme mediates dynamic spectral tuning of the entire visual system by controlling the balance of vitamin A1 and A2 in the eye.
Xenobiotica; the fate of foreign compounds in biological systems, Jan 6, 2015
1. The polycyclic hydrocarbons (PAHs), pyrene, 1-hydroxypyrene, 1-nitropyrene and 1-acetylpyrene,... more 1. The polycyclic hydrocarbons (PAHs), pyrene, 1-hydroxypyrene, 1-nitropyrene and 1-acetylpyrene, were found to induce Type I binding spectra with human cytochrome P450 (P450) 2A13 and were converted to various mono- and di-oxygenated products by this enzyme. 2. Pyrene was first oxidized by P450 2A13 to 1-hydroxypyrene which was further oxidized to di-oxygenated products, i.e. 1,8- and 1,6-dihydroxypyrene. Of five other human P450s examined, P450 1B1 catalyzed pyrene oxidation to 1-hydroxypyrene at a similar rate to P450 2A13 but was less efficient in forming dihydroxypyrenes. P450 2A6, a related human P450 enzyme, which did not show any spectral changes with these four PAHs, showed lower activities in oxidation of these compounds than P450 2A13. 3. 1-Nitropyrene and 1-acetylpyrene were also found to be efficiently oxidized by P450 2A13 to several oxygenated products, based on mass spectrometry analysis. 4. Molecular docking analysis supported preferred orientations of pyrene and it...
Journal of Biological Chemistry, 2015
Background: Mammals synthesize endogenous morphine; the enzyme catalyzing thebaine O 6-demethylat... more Background: Mammals synthesize endogenous morphine; the enzyme catalyzing thebaine O 6-demethylation, a key late step, is uncharacterized. Results: Human cytochromes P450 (P450) 3A4 and 3A5 catalyzed thebaine O 6-demethylation; the P450 3A-selective drug ketoconazole inhibited the reaction in human liver microsomes and rat brain homogenates. Conclusion: P450s 3A4 and 3A5 catalyze thebaine O 6-demethylation in humans. Significance: Enzymes catalyzing all oxidations in the latter steps of mammalian morphine biosynthesis have been identified.
The Journal of biological chemistry, Jan 22, 2014
Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progeste... more Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s: zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the 2-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1, but only in the case of Prog. X-ray crystal s...
Chemico-Biological Interactions, 2012
Nicotine is the primary addictive agent in tobacco products and is metabolized in humans by CYP2A... more Nicotine is the primary addictive agent in tobacco products and is metabolized in humans by CYP2A6. Decreased CYP2A6 activity has been associated with decreased smoking. The extrahepatic enzyme, CYP2A13 (94% identical to CYP2A6) also catalyzes the metabolism of nicotine, but is most noted for its role in the metabolic activation of the tobacco specific lung carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study, the inhibition and potential inactivation of CYP2A6 and CYP2A13 by two tobacco constituents, 1-methyl-4-(3pyridinyl) pyrrole (β-nicotyrine) and (−)-menthol were characterized and compared to the potent mechanism based inactivator of CYP2A6, menthofuran. The effect of these compounds on CYP2A6 and CYP2A13 activity was significantly different. (−)-Menthol was a more efficient inhibitor of CYP2A13 than of CYP2A6 (K I , 8.2 μM and 110 μM, respectively). β-nicotyrine was a potent inhibitor of CYP2A13 (K I , 0.17 μM). Neither menthol nor β-nicotyrine were inactivators of CYP2A13. Whereas, β-nicotyrine was a mechanism based inactivator of CYP2A6 (K I(inact) , 106 μM, k inact was 0.61 min −1). Similarly, menthofuran, a potent mechanism based inactivator of CYP2A6 did not inactivate CYP2A13. Menthofuran was an inhibitor of CYPA13 (K I , 1.24 μM). The inactivation of CYP2A6 by either β-nicotyrine or menthofuran was not due to modification of the heme and was likely due to modification of the apo-protein. These studies suggest that βnicotyrine, but not menthol may influence nicotine and NNK metabolism in smokers.
Journal of Biological Chemistry, 2014
Background: Fish (and human) P450 17A1 catalyze both steroid 17␣-hydroxylation and 17␣,20-lyase r... more Background: Fish (and human) P450 17A1 catalyze both steroid 17␣-hydroxylation and 17␣,20-lyase reactions. A second fish P450, 17A2 (51% identical), catalyzes only 17␣-hydroxylation. Results: Crystal structures of zebrafish P450 17A1 and 17A2 and human P450 17A1 are very similar. Conclusion: In kinetic analysis, the two-step oxidation of progesterone is more distributive than for pregnenolone. Significance: Small structural differences are associated with activities of the two fish P450s. Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals, a single enzyme, P450 17A1, catalyzes both 17␣-hydroxylation and a subsequent 17␣,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17␣-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17␣hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b 5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulsechase and kinetic spectral experiments and modeling established that the two-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17␣-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1 but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and with Prog for P450 17A2. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116-119) showed only a few differences near the active site, despite only ϳ50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternative ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity.
The Journal of Biological Chemistry, Feb 10, 2006
DNA gyrase is the only topoisomerase that can introduce negative supercoils into DNA. It is thoug... more DNA gyrase is the only topoisomerase that can introduce negative supercoils into DNA. It is thought that the binding of conventional type II topoisomerases, including topoisomerase IV, to DNA takes place at the catalytic domain across the DNA gate, whereas DNA gyrase binds to DNA not only at the amino-terminal catalytic domain but also at the carboxyl-terminal domain (CTD) of the GyrA subunit. The binding of the GyrA CTD to DNA allows gyrase to wrap DNA around itself and catalyze the supercoiling reaction. Recent structural studies, however, have revealed striking similarities between the GyrA CTD and the ParC CTD, as well as the ability of the ParC CTD to bind and bend DNA. Thus, the molecular basis of gyrase-mediated wrapping of DNA needs to be reexamined. Here, we have conducted a mutational analysis to determine the role of the "GyrA-box," a 7-amino acid-long motif unique to the GyrA CTD, in determining the DNA binding mode of gyrase. Either a deletion of the entire GyrA-box or substitution of the GyrA-box with 7 Ala residues abolishes the ability of gyrase to wrap DNA around itself and catalyze the supercoiling reaction. However, these mutations do not affect the relaxation and decatenation activities of gyrase. Thus, the presence of a GyrA-box allows gyrase to wrap DNA and catalyze the supercoiling reaction. The consequence of the loss of the GyrA-box during evolution of bacterial type II topoisomerases is discussed.
Drug metabolism letters
Timolol is clinically administered topically (ocular) to reduce intraocular pressure and treat op... more Timolol is clinically administered topically (ocular) to reduce intraocular pressure and treat open-angle glaucoma. Ocular administration of timolol in low doses (0.5% w/v in the form of eye drops) has led to challenges for in vivo metabolite identification. An understanding of drug metabolism in the eye is important for clinical ocular therapeutics and potential drug candidates. We aimed to investigate the metabolism of timolol in rat ocular and liver S9 fractions, as well as rat ocular tissue and plasma following a 0.5% topical (ocular) dose of timolol. We explored the potential in vitro metabolic bioactivation in the eye/liver by conducting trapping studies for putative aldehyde and iminium ion intermediates that may arise from the morpholine functionality. Oxidative metabolism of timolol to its major metabolite (M4) in ocular S9 and recombinant rat cytochrome P450 (CYP) isoforms supports the possible role of rat ocular CYP2D2, 2D4, and/or 2D18. Observation of N-acetyl-timolol (M...
Journal of Pharmaceutical Sciences
Nicotine, the primary addictive compound in cigarettes, is metabolized in humans by cytochrome P4... more Nicotine, the primary addictive compound in cigarettes, is metabolized in humans by cytochrome P450 2A enzymes. The hepatic enzyme responsible for the metabolism of nicotine in smokers is P450 2A6. P450 2A13, which shares 94% primary sequence homology with P450 2A6, also catalyzes the metabolism of nicotine and is present in the lung. Loss of P450 2A activity is correlated with modified smoking behavior and addiction. Inhibition of these enzymes decreases nicotine metabolism and may be of benefit in smoking cessation. This thesis investigates tobacco-related molecules that may impact P450 2A function and is presented in three parts. In the first, the potential inhibitory potency of (-)-menthol, (R)-(+)-menthofuran, and -nicotyrine of both P450s 2A6 and 2A13 are investigated. All three compounds inhibit P450 2A6 and 2A13 activity. In addition, menthofuran and -nicotyrine mediate mechanism-based inactivation of P450 2A6 but not 2A13. Second, the P450 2A6 and P450 2A13-mediated metabolism of -nicotyrine is studied and three metabolites are identified. -nicotyrine is readily turned over by both P450 2A6 and P450 2A13 as indicated by the calculated K m (4.4µM and 5.0µM, respectively) and V max (21 and 37 pmol product/min/pmol, respectively) values. Also in the second section, -nicotyrine is shown to be a metabolite of P450 2A6-mediated nicotine metabolism. In the last section, attempts to identify a covalent adduct that would result from menthofuran or -nicotyrine-mediated inactivation are presented, but these are largely unsuccessful.
FEBS Letters, 2016
In humans, a considerable fraction of the retinoid pool in skin is derived from vitamin A2 (all-t... more In humans, a considerable fraction of the retinoid pool in skin is derived from vitamin A2 (all-trans 3,4-dehydroretinal). Vitamin A2 may be locally generated by keratinocytes, which can convert vitamin A1 (all-trans retinol) into vitamin A2 in cell culture. We report that human cytochrome P450 (hP450) 27C1, a previously 'orphan' enzyme, can catalyze this reaction. Purified recombinant hP450 27C1 bound and desaturated all-trans retinol, retinal, and retinoic acid, as well as 11-cis retinal. Although the physiological role of 3,4-dehydroretinoids in humans is unclear, we have identified hP450 27C1 as an enzyme capable of efficiently mediating their formation. This article is protected by copyright. All rights reserved.
Current Biology, 2015
Some vertebrate species have evolved means of extending their visual sensitivity beyond the range... more Some vertebrate species have evolved means of extending their visual sensitivity beyond the range of human vision. One mechanism of enhancing sensitivity to long-wavelength light is to replace the 11-cis retinal chromophore in photopigments with 11-cis 3,4-didehydroretinal. Despite over a century of research on this topic, the enzymatic basis of this perceptual switch remains unknown. Here, we show that a cytochrome P450 family member, Cyp27c1, mediates this switch by converting vitamin A1 (the precursor of 11-cis retinal) into vitamin A2 (the precursor of 11-cis 3,4didehydroretinal). Knockout of cyp27c1 in zebrafish abrogates production of vitamin A2, eliminating the animal's ability to red-shift its photoreceptor spectral sensitivity and reducing its ability to see and respond to near-infrared light. Thus, the expression of a single enzyme mediates dynamic spectral tuning of the entire visual system by controlling the balance of vitamin A1 and A2 in the eye.
Xenobiotica; the fate of foreign compounds in biological systems, Jan 6, 2015
1. The polycyclic hydrocarbons (PAHs), pyrene, 1-hydroxypyrene, 1-nitropyrene and 1-acetylpyrene,... more 1. The polycyclic hydrocarbons (PAHs), pyrene, 1-hydroxypyrene, 1-nitropyrene and 1-acetylpyrene, were found to induce Type I binding spectra with human cytochrome P450 (P450) 2A13 and were converted to various mono- and di-oxygenated products by this enzyme. 2. Pyrene was first oxidized by P450 2A13 to 1-hydroxypyrene which was further oxidized to di-oxygenated products, i.e. 1,8- and 1,6-dihydroxypyrene. Of five other human P450s examined, P450 1B1 catalyzed pyrene oxidation to 1-hydroxypyrene at a similar rate to P450 2A13 but was less efficient in forming dihydroxypyrenes. P450 2A6, a related human P450 enzyme, which did not show any spectral changes with these four PAHs, showed lower activities in oxidation of these compounds than P450 2A13. 3. 1-Nitropyrene and 1-acetylpyrene were also found to be efficiently oxidized by P450 2A13 to several oxygenated products, based on mass spectrometry analysis. 4. Molecular docking analysis supported preferred orientations of pyrene and it...
Journal of Biological Chemistry, 2015
Background: Mammals synthesize endogenous morphine; the enzyme catalyzing thebaine O 6-demethylat... more Background: Mammals synthesize endogenous morphine; the enzyme catalyzing thebaine O 6-demethylation, a key late step, is uncharacterized. Results: Human cytochromes P450 (P450) 3A4 and 3A5 catalyzed thebaine O 6-demethylation; the P450 3A-selective drug ketoconazole inhibited the reaction in human liver microsomes and rat brain homogenates. Conclusion: P450s 3A4 and 3A5 catalyze thebaine O 6-demethylation in humans. Significance: Enzymes catalyzing all oxidations in the latter steps of mammalian morphine biosynthesis have been identified.
The Journal of biological chemistry, Jan 22, 2014
Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progeste... more Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s: zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the 2-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1, but only in the case of Prog. X-ray crystal s...
Chemico-Biological Interactions, 2012
Nicotine is the primary addictive agent in tobacco products and is metabolized in humans by CYP2A... more Nicotine is the primary addictive agent in tobacco products and is metabolized in humans by CYP2A6. Decreased CYP2A6 activity has been associated with decreased smoking. The extrahepatic enzyme, CYP2A13 (94% identical to CYP2A6) also catalyzes the metabolism of nicotine, but is most noted for its role in the metabolic activation of the tobacco specific lung carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study, the inhibition and potential inactivation of CYP2A6 and CYP2A13 by two tobacco constituents, 1-methyl-4-(3pyridinyl) pyrrole (β-nicotyrine) and (−)-menthol were characterized and compared to the potent mechanism based inactivator of CYP2A6, menthofuran. The effect of these compounds on CYP2A6 and CYP2A13 activity was significantly different. (−)-Menthol was a more efficient inhibitor of CYP2A13 than of CYP2A6 (K I , 8.2 μM and 110 μM, respectively). β-nicotyrine was a potent inhibitor of CYP2A13 (K I , 0.17 μM). Neither menthol nor β-nicotyrine were inactivators of CYP2A13. Whereas, β-nicotyrine was a mechanism based inactivator of CYP2A6 (K I(inact) , 106 μM, k inact was 0.61 min −1). Similarly, menthofuran, a potent mechanism based inactivator of CYP2A6 did not inactivate CYP2A13. Menthofuran was an inhibitor of CYPA13 (K I , 1.24 μM). The inactivation of CYP2A6 by either β-nicotyrine or menthofuran was not due to modification of the heme and was likely due to modification of the apo-protein. These studies suggest that βnicotyrine, but not menthol may influence nicotine and NNK metabolism in smokers.
Journal of Biological Chemistry, 2014
Background: Fish (and human) P450 17A1 catalyze both steroid 17␣-hydroxylation and 17␣,20-lyase r... more Background: Fish (and human) P450 17A1 catalyze both steroid 17␣-hydroxylation and 17␣,20-lyase reactions. A second fish P450, 17A2 (51% identical), catalyzes only 17␣-hydroxylation. Results: Crystal structures of zebrafish P450 17A1 and 17A2 and human P450 17A1 are very similar. Conclusion: In kinetic analysis, the two-step oxidation of progesterone is more distributive than for pregnenolone. Significance: Small structural differences are associated with activities of the two fish P450s. Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals, a single enzyme, P450 17A1, catalyzes both 17␣-hydroxylation and a subsequent 17␣,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17␣-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17␣hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b 5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulsechase and kinetic spectral experiments and modeling established that the two-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17␣-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1 but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and with Prog for P450 17A2. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116-119) showed only a few differences near the active site, despite only ϳ50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternative ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity.
The Journal of Biological Chemistry, Feb 10, 2006
DNA gyrase is the only topoisomerase that can introduce negative supercoils into DNA. It is thoug... more DNA gyrase is the only topoisomerase that can introduce negative supercoils into DNA. It is thought that the binding of conventional type II topoisomerases, including topoisomerase IV, to DNA takes place at the catalytic domain across the DNA gate, whereas DNA gyrase binds to DNA not only at the amino-terminal catalytic domain but also at the carboxyl-terminal domain (CTD) of the GyrA subunit. The binding of the GyrA CTD to DNA allows gyrase to wrap DNA around itself and catalyze the supercoiling reaction. Recent structural studies, however, have revealed striking similarities between the GyrA CTD and the ParC CTD, as well as the ability of the ParC CTD to bind and bend DNA. Thus, the molecular basis of gyrase-mediated wrapping of DNA needs to be reexamined. Here, we have conducted a mutational analysis to determine the role of the "GyrA-box," a 7-amino acid-long motif unique to the GyrA CTD, in determining the DNA binding mode of gyrase. Either a deletion of the entire GyrA-box or substitution of the GyrA-box with 7 Ala residues abolishes the ability of gyrase to wrap DNA around itself and catalyze the supercoiling reaction. However, these mutations do not affect the relaxation and decatenation activities of gyrase. Thus, the presence of a GyrA-box allows gyrase to wrap DNA and catalyze the supercoiling reaction. The consequence of the loss of the GyrA-box during evolution of bacterial type II topoisomerases is discussed.