ABSTRACT
At least two substitutions were made at each of five amino acid residues in rat cytochrome P450 2B1 that align to residues of known importance in other P450s. The mutants were histidine tagged for purification from Escherichia coli, and the proteins were assessed for testosterone and 7-alkoxycoumarin oxidation. Alteration of each of the sites studied, Phe-115, Ser-294, Phe-297, Ala-298, and Leu-362, was found to affect overall enzyme activity or the metabolite profile. In particular, most of the mutants, excluding F297A, A298G, and L362F, exhibited significantly altered ratios of 16alpha-hydroxytestosterone:16beta-hydroxytestosterone, with the most dramatic alteration being displayed by A298V. Four 7-butoxycoumarin metabolites were produced by CYP2B1, of which two, 7-hydroxycoumarin and 7-(3-hydroxybutoxy)coumarin, were formed at nearly equal rates. Several mutants, F115A, F297A, F297I, and A298V, exhibited an increased predominance of one of the metabolites. The results from this study illustrate the conservation of functionally important residues across P450 subfamilies and families.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Coumarins/chemistry , Coumarins/metabolism , Cytochrome P-450 CYP2B1/chemistry , Cytochrome P-450 CYP2B1/metabolism , Steroids/metabolism , Amino Acid Substitution , Animals , Binding Sites , Cytochrome P-450 CYP2B1/genetics , Cytochrome P-450 CYP2B1/isolation & purification , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/isolation & purification , Cytochrome P-450 Enzyme System/metabolism , Hydroxylation , Models, Molecular , Mutation , Oxidants/metabolism , Oxidation-Reduction , Protein Conformation , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Steroid Hydroxylases/chemistry , Steroid Hydroxylases/genetics , Steroid Hydroxylases/isolation & purification , Steroid Hydroxylases/metabolism , Steroids/chemistry , Substrate Specificity , Testosterone/chemistry , Testosterone/metabolismABSTRACT
Phenylalanine and/or tryptophan scanning mutagenesis was performed at 15 sites within CYP3A4 proposed to be involved in substrate specificity or cooperativity. The sites were chosen on the basis of previous studies or from a comparison with the structure of P450(eryF) containing two molecules of androstenedione. The function of the 25 mutants was assessed in a reconstituted system using progesterone, testosterone, 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), and alpha-naphthoflavone (ANF) as substrates. CYP3A4 wild type displayed sigmoidal kinetics of ANF 5,6-oxide formation and 7-BFC debenzylation. Analysis of 12 mutants with significant steroid hydroxylase activity showed a lack of positive correlation between ANF oxidation and stimulation of progesterone 6beta-hydroxylation by ANF, indicating that ANF binds at two sites within CYP3A4. 7-BFC debenzylation was stimulated by progesterone and ANF, and 7-BFC did not inhibit testosterone or progesterone 6beta-hydroxylation. Correlational analysis showed no relationship between 7-BFC debenzylation and either progesterone or testosterone 6beta-hydroxylation. These data are difficult to explain with a two-site model of CYP3A4 but suggest that three subpockets exist within the active site. Interestingly, classification of the mutants according to their ability to oxidize the four substrates utilized in this study suggested that substrates do bind at preferred locations in the CYP3A4 binding pocket.
Subject(s)
Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/metabolism , Phenylalanine , Tryptophan , Amino Acid Sequence , Amino Acid Substitution , Base Sequence , Binding Sites , Cloning, Molecular , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/genetics , DNA Primers , Escherichia coli , Humans , Kinetics , Liver/enzymology , Mixed Function Oxygenases/genetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Progesterone/metabolism , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
Over the past decade, site-directed mutagenesis has become an essential tool in the study of mammalian cytochrome P450 structure-function relationships. Residues affecting substrate specificity, cooperativity, membrane localization, and interactions with redox partners have been identified using a combination of amino-acid sequence alignments, homology modeling, chimeragenesis, and site-directed mutagenesis. As homology modeling and substrate docking technology continue to improve, the ability to predict more precise functions for specific residues will also advance, making it possible to utilize site-directed mutagenesis to test these predictions. Future studies will employ site-directed mutagenesis to learn more about cytochrome P450 substrate access channels, to define the role of residues that do not lie within substrate recognition sites, to engineer additional soluble forms of microsomal cytochromes P450 for x-ray crystallography, and to engineer more efficient enzymes for drug activation and/or bioremediation.
Subject(s)
Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/physiology , Mutagenesis, Site-Directed , Animals , Binding Sites , Forecasting , Humans , Models, Molecular , Molecular Structure , Polymorphism, Genetic , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Structure-Activity Relationship , Substrate SpecificityABSTRACT
The genetic component of the inter-individual variability in CYP3A4 activity has been estimated to be between 60% and 90%, but the underlying genetic factors remain largely unknown. A study of 213 Middle and Western European DNA samples resulted in the identification of 18 new CYP3A4 variants, including eight protein variants. A total of 7.5% of the population studied was found to be heterozygous for one of these variants. In a bacterial heterologous expression system, two mutants, R130Q and P416L, did not result in detectable P450 holoprotein. One mutant, T363M, expressed at significantly lower levels than wild-type CYP3A4. G56D, V170I, D174H and M445T were not significantly different when compared with wild-type CYP3A4 in expression or steroid hydroxylase activity. L373F displayed a significantly altered testosterone metabolite profile and a four-fold increase in the Km value for 1'-OH midazolam formation. The results suggest a limited contribution of CYP3A4 protein variants to the inter-individual variability of CYP3A4 activity in Caucasians. Some variants may, however, play a role in the atypical response to drugs or altered sensitivity to carcinogens.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Isoenzymes/genetics , Mixed Function Oxygenases/genetics , Base Sequence , Cytochrome P-450 CYP3A , DNA Primers , Humans , Mutagenesis , Polymerase Chain ReactionABSTRACT
The major human liver drug-metabolizing cytochrome P450 enzymes P450 3A4 and P450 3A5 share >85% amino acid sequence identity yet exhibit different regioselectivity toward aflatoxin B(1) (AFB(1)) biotransformation [Gillam et al. (1995) Arch. Biochem. Biophys. 317, 74-384]. P450 3A4 prefers AFB1 3alpha-hydroxylation, which detoxifies and subsequently eliminates the hepatotoxin, over AFB1 exo-8,9-oxidation. P450 3A5, on the other hand, is a relatively sluggish 3alpha-hydroxylase and converts AFB(1) predominantly to the genotoxic exo-8,9-epoxide. Using a combination of approaches (sequence alignment, homology modeling and site-directed mutagenesis), we have previously identified several divergent residues in four of the six putative substrate recognition sites (SRSs) of P450 3A4, which when replaced individually with the corresponding amino acid of P450 3A5, resulted in a significant switch of the characteristic P450 3A4 AFB(1) regioselectivity toward that of P450 3A5 [Wang et al. (1998) Biochemistry 37, 12536-12545]. In particular, residues N206 and L210 in SRS-2 were found to be critical for AFB(1) detoxification via 3alpha-hydroxylation, and the corresponding mutants N206S and L210F most closely mimicked P450 3A5, not only in its regioselectivity of AFB(1) metabolism but also in its overall functional capacity. We have now further explored the plausible reasons for such relative inactivity of the SRS-2 mutants by examining N206S and additional mutants (L210A, L211F, L211A, and N206E) and found that the dramatically lowered activities of the N206S mutant are accompanied by a loss of cooperativity of AFB(1) oxidation. Molecular dynamics analyses with an existing P450 3A4 homology model [Szklarz and Halpert (1997) J. Comput. Aided Mol. Des. 11, 265] suggested that N206 (helix F) interacts with E244 (helix G), creating a salt bridge that stabilizes the protein structure and/or defines the active site cavity. To examine this possibility, several E244 mutants (E244A, V, N, S) were tested, of which E244S was the most notable for its relatively greater impairment of P450 3A4-dependent AFB(1) 3alpha-hydroxylation. However, the results with these E244 mutants failed to validate the N206-E244 interaction predicted from these molecular dynamics analyses. Collectively, our findings to date have led us to reconsider our original interpretations and to reexamine them in the light of AFB(1) molecular modeling analyses with a newly refined P450 3A4 homology model. These analyses predicted that F304 in SRS-4 (I-helix) plays a pivotal role in AFB(1) binding at the active site in either orientation leading to 3alpha- or exo-8,9-oxidation. Consistent with this prediction, conversion of F304 to Ala abolished P450 3A4-dependent AFB(1) 3alpha-hydroxylation and exo-8,9-oxidation.
Subject(s)
Aflatoxin B1/metabolism , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Mutagenesis, Site-Directed/genetics , Phenylalanine/metabolism , Aflatoxin B1/chemistry , Amino Acid Motifs/physiology , Binding Sites/physiology , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/chemistry , Escherichia coli/genetics , Hydroxylation , Inactivation, Metabolic/physiology , Mixed Function Oxygenases/chemistry , Models, Molecular , Oxidation-Reduction , Phenylalanine/genetics , Sequence Alignment , Sequence Homology , Substrate SpecificityABSTRACT
The RACE amplification technology was used on a novel CYP3A-like exon 1 sequence detected during the reverse transcriptase/polymerase chain reaction analysis of human CYP3A gene expression. This resulted in the identification of cDNAs encompassing the complete coding sequence of a new member of the CYP3A gene subfamily, CYP3A43. Interestingly, the majority of the cDNAs identified were characterized by alternative splicing events such as exon skipping and complete or partial intron inclusion. CYP3A43 expression was detected in liver, kidney, pancreas, and prostate. The amino acid sequence is 75% identical to that of CYP3A4 and CYP3A5 and 71% identical to CYP3A7. CYP3A43 differs from CYP3A4 at six amino acid residues, found within the putative substrate recognition sites of CYP3A4, that are known to be determinants of substrate selectivity. The N terminus of CYP3A43 was modified for efficient expression of the protein in Escherichia coli, and a 6X histidine tag was added at the C terminus to facilitate purification. CYP3A43 gave a reduced carbon monoxide difference spectra with an absorbance maximum at 450 nm. The level of heterologous expression was significantly lower than that observed for CYP3A4 and CYP3A5. Immunoblot analyses revealed that CYP3A43 comigrates with CYP3A4 in polyacrylamide gel electrophoresis but does separate from CYP3A5. Monooxygenase assays were performed under a variety of conditions, several of which yielded reproducible, albeit low, testosterone hydroxylase activity. The findings from this study demonstrate that there is a novel CYP3A member expressed in human tissues, although its relative contribution to drug metabolism has yet to be ascertained.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/genetics , DNA, Complementary/analysis , Amino Acid Sequence , Blotting, Western , Cloning, Molecular , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/isolation & purification , Cytochrome P-450 Enzyme System/metabolism , DNA Primers , Escherichia coli , Exons , Humans , Mixed Function Oxygenases/chemistry , Molecular Sequence Data , Molecular Weight , Oxidoreductases, N-Demethylating/chemistry , Polymerase Chain Reaction , Sequence Homology, Amino AcidABSTRACT
The structural basis of cooperativity of progesterone hydroxylation catalyzed by human cytochrome P450 3A4 has been investigated. A recent study suggested that substitution of larger side chains at positions Leu-211 and Asp-214 partially mimics the action of effector by reducing the size of the active site. Based on predictions from molecular modeling that Phe-304 in the highly conserved I helix is involved in both effector and substrate binding, a tryptophan residue was substituted at this position. The purified F304W mutant displayed hyperbolic progesterone hydroxylase kinetics, indicating a lack of homotropic cooperativity. However, the mutant remained responsive to stimulation by alpha-naphthoflavone, exhibiting a 2-fold decrease in the K(m) value for progesterone 6beta-hydroxylation in the presence of 25 microM effector. Combining substitutions to yield the triple mutant L211F/D214E/F304W maintained the V(max) and decreased the K(m) for progesterone 6beta-hydroxylation, minimized stimulation by alpha-naphthoflavone, and decreased the rate of alpha-naphthoflavone oxidation to one-eighth of the wild type. Interestingly, the DeltaA(max) for spectral binding of alpha-naphthoflavone was unaltered in L211F/D214E/F304W. Overall, the results suggest that progesterone and alpha-naphthoflavone are oxidized at separate locations within the P450 3A4 binding pocket, although both substrates appear to have equal access to the reactive oxygen.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/metabolism , Mixed Function Oxygenases/metabolism , Phenylalanine/metabolism , Benzoflavones/metabolism , Binding Sites , Cloning, Molecular , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/genetics , Humans , Hydroxylation , Kinetics , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/genetics , Models, Molecular , Mutation/genetics , Mutation/physiology , Oxidation-Reduction , Phenylalanine/chemistry , Progesterone/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Steroid Hydroxylases/metabolism , Substrate SpecificityABSTRACT
The structural basis for functional differences between human cytochrome P-450 2B6 and rat 2B1 was investigated. An amino acid sequence alignment predicted the location of 2B6 substrate recognition site (SRS) residues. Ten residues within these SRSs unique to 2B6 compared with 2B1, 2B4, and 2B11 were chosen for mutagenesis. Two additional sites that differ between 2B6 and 2B1 and are known to have a role in 2B1 substrate specificity were also mutated. The 2B6 mutants were expressed in Spodoptera frugiperda cells and characterized using the 2B6-specific substrate RP 73401 [3-cyclopentyloxy-N-(3,5-dichloro-4-pyridyl)-4-methoxybenzamide], the 2B1-selective substrate androstenedione, and the common substrate 7-ethoxy-4-trifluoromethylcoumarin. Mutants F107I and L363V exhibited decreased RP 73401 hydroxylation but retained most of the wild-type level of 2B6 7-ethoxy-4-trifluoromethylcoumarin O-deethylase activity. In addition, SRS exchanges were studied in which the amino acid sequence of 2B6 SRSs was converted to the sequence of 2B1. Each of these constructs, having two to seven substitutions, expressed at levels similar to 2B6 but did not acquire significant androstenedione hydroxylase activity. Docking of RP 73401 into the active site of a 2B6 homology model suggested a direct interaction with residue L363 but not with F107. Findings from this study suggest that 1) residues F107 and L363 are necessary for 2B6 RP 73401 hydroxylase activity, 2) 2B6 is able to tolerate multiple SRS substitutions without compromising protein expression levels or protein stability, and 3) conferring androstenedione hydroxylase function to cytochrome P-450 2B6 is more complex than altering a single SRS.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Oxidoreductases, N-Demethylating/genetics , Oxidoreductases, N-Demethylating/metabolism , Animals , Baculoviridae/genetics , Binding Sites/genetics , Cytochrome P-450 CYP2B6 , Cytochrome P-450 Enzyme System/biosynthesis , Cytochrome P-450 Enzyme System/chemistry , Enzyme Activation/genetics , Humans , Kinetics , Oxidoreductases, N-Demethylating/biosynthesis , Oxidoreductases, N-Demethylating/chemistry , Rats , Sequence Alignment , Sequence Homology, Amino Acid , Software , Spodoptera/genetics , Spodoptera/virology , Structure-Activity Relationship , Substrate SpecificityABSTRACT
RPR 106541 (20R-16alpha,17alpha-[butylidenebis(oxy)]-6al pha, 9alpha-difluoro-11beta-hydroxy-17beta-(methylthio)androst a-4-en-3-one) is an airway-selective steroid developed for the treatment of asthma. Two metabolites produced by human liver microsomes were identified as R- and S-sulfoxide diastereomers based on liquid chromatography/mass spectrometry analysis, proton nuclear magnetic resonance, and cochromatography with standards. Sulfoxide formation was determined to be cytochrome P-450 (CYP) 3A4-dependent by correlation with CYP3A4-marker nifedipine oxidase activity, inhibition by cyclosporin A and troleandomycin, and inhibition of R- (70%) and S- (64%) sulfoxide formation by anti-3A antibody. Expressed CYP2C forms catalyzed RPR 106541 sulfoxidation; however, other phenotyping approaches failed to confirm the involvement of CYP2C forms in these reactions in human liver microsomes. Expressed CYP3A4 catalyzed the formation of the sulfoxide diastereomers in a 1:1 ratio, whereas CYP3A5 displayed stereoselectivity for formation of the S-diastereomer. The high rate of sulfoxidation by CYP3A4 and the blockage of oxidative metabolism at the electronically favored 6beta-position provided advantages for RPR 106541 over other substrates as an active site probe of CYP3A4. Therefore, oxidation of RPR 106541 by various CYP3A4 substrate recognition site (SRS) mutants was assessed. In SRS-4, A305V and F304A showed dramatically reduced rates of R-diastereomer formation (83 and 64% decreases, respectively), but S-diastereomer formation was affected to a lesser extent. A370V (SRS-5) showed decreased formation of the R-sulfoxide (52%) but increased formation of the S-diastereomer. In the SRS-2 region, the most dramatic change in sulfoxide ratios was observed for L210A. In conclusion, the structure of RPR 106541 imposes specific constraints on enzyme binding and activity and thus represents an improved CYP3A4 probe substrate.
Subject(s)
Androstenes/metabolism , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/genetics , Mixed Function Oxygenases/antagonists & inhibitors , Mixed Function Oxygenases/genetics , Androstenes/chemistry , Animals , Chromatography, High Pressure Liquid , Computer Simulation , Crystallography, X-Ray , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/chemistry , Dogs , Humans , Mass Spectrometry , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Mixed Function Oxygenases/chemistry , Models, Molecular , Mutagenesis, Site-Directed , Mutation , Rats , Rats, Sprague-Dawley , Stereoisomerism , Structure-Activity Relationship , Sulfoxides/metabolismABSTRACT
This article is a report on a symposium held at Experimental Biology '98 in San Francisco, California. Recent developments in site-directed mutagenesis, computer-modeling, and mechanistic analysis of cytochromes P450 and flavin-containing monooxygenases are described. A unifying theme is the elaboration of general approaches for understanding and predicting the function of individual forms of these enzymes. A related goal is the production of soluble forms of mammalian cytochromes P450 for X-ray crystallography.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Flavins/metabolism , Mixed Function Oxygenases/metabolism , Pharmaceutical Preparations/metabolism , Animals , Cytochrome P-450 Enzyme System/chemistry , Humans , Structure-Activity RelationshipABSTRACT
Sequence alignment of human cytochrome P450 3A4 with bacterial enzymes of known structure has provided a basis from which to predict residues involved in substrate oxidation. Substitutions were made at four residues (I301, F304, A305, and T309) predicted to be located within the highly conserved substrate recognition site 4. Site-directed mutants engineered to contain carboxy-terminal histidine tags were expressed in Escherichia coli and purified on a metal affinity column. The integrity of each protein was assessed by SDS-polyacrylamide gel electrophoresis and immunoblotting. Functional analysis was performed using progesterone and testosterone as substrates and alpha-naphthoflavone as an activator. In testosterone hydroxylase assays, all of the mutants displayed rates of total product formation similar to wild-type 3A4, with several mutants showing small differences in specific products formed. However, with progesterone as the substrate, mutants F304A, A305V, and T309A exhibited altered product ratios and/or changes in the rates of product formation. F304A and A305V also displayed altered flavonoid stimulation that resulted in product ratios dramatically different from wild-type 3A4. Therefore, the kinetics of progesterone hydroxylation of these mutants and the wild-type enzyme were further assessed, and the data were analyzed with the Hill equation. Results with wild-type 3A4 and F304A indicated that at high progesterone concentrations, hydroxylation rates and product ratios are independent of the presence of alpha-NF. This suggests that progesterone may be equivalent to alpha-NF as an activator. In contrast, A305V exhibited autoactivation by progesterone but inhibition by alpha-NF.
Subject(s)
Benzoflavones/pharmacology , Cytochrome P-450 Enzyme System/chemistry , Mixed Function Oxygenases/chemistry , Steroid Hydroxylases/metabolism , Amino Acid Sequence , Binding Sites/genetics , Conserved Sequence , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/physiology , Enzyme Activation , Humans , Hydroxylation , Kinetics , Mixed Function Oxygenases/physiology , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed/genetics , Progesterone/metabolism , Recombinant Proteins/metabolism , Sequence Alignment , Testosterone/metabolismABSTRACT
The high level of cross-linking found in Staphylococcus aureus peptidoglycan is dependent on the low-molecular-weight penicillin-binding protein PBP4. Recently, the PBP4 gene, pbpD, was cloned and shown to be adjacent to and divergently transcribed relative to the putative ABC-type transporter gene, abcA. Disruption of abcA (in strain KB400) was previously shown to result in heightened resistance to several antibiotics known to interact with PBP4, suggesting that the regulation of pbpD is affected by abcA. In this report, this hypothesis was confirmed by use of a Northern (RNA) blot analysis which revealed increased accumulation of pbpD-specific transcripts in KB400 compared to that in the wild-type strain, 8325-4. By using reverse-phase high-performance liquid chromatography to examine the structure of the peptidoglycan, it was demonstrated that the increased expression of pbpD resulted in an increased level of peptidoglycan cross-linking in the staphylococcal cell wall. Promoter fusion studies demonstrated that the abcA mutation caused approximately 7-fold and 100-fold increases in pbpD and abcA promoter activities, respectively. Primer extension experiments revealed that these genes have long, untranslated leader sequences that result in a transcriptional overlap of 80 bp. Interestingly, deletion of a 26-bp region containing an inverted repeat sequence resulted in the loss of expression from both the abcA and the pbpD promoters. These data provide evidence that abcA and pbpD are under the control of a common regulatory mechanism that may involve the transport function of the abcA gene product.
Subject(s)
ATP-Binding Cassette Transporters , Carrier Proteins/genetics , Dioxygenases , Gene Expression Regulation, Bacterial/genetics , Hexosyltransferases , Muramoylpentapeptide Carboxypeptidase/genetics , Peptidyl Transferases , Staphylococcus aureus/genetics , Transcription, Genetic/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Base Sequence , Catechol 2,3-Dioxygenase , Cell Wall/chemistry , Genes, Bacterial/genetics , Molecular Sequence Data , Mutation , Oxygenases/genetics , Oxygenases/metabolism , Penicillin-Binding Proteins , Peptidoglycan/chemistry , Phylogeny , Promoter Regions, Genetic/genetics , RNA, Bacterial/biosynthesis , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Recombinant Fusion Proteins , Repetitive Sequences, Nucleic AcidABSTRACT
A region of the Staphylococcus aureus chromosome has been isolated that contains the gene encoding penicillin-binding protein 4 (PBP4), as well as abcA, a gene that encodes a protein with strong sequence similarity to the ABC transporter family of proteins. A disruption in abcA by Campbell-type integration results in cells that display an increased resistance to methicillin and cefoxitin, two antibiotics known to interact with low-molecular-weight PBPs. Based on these observations, a potential regulatory link between these two genes is discussed.