Purification and characterization of macrolide 2′-phosphotransferase type II from a strain of Escherichia coli highly resistant to macrolide antibiotics

Kono, Megumi

doi:10.1016/0378-1097(92)90369-y

Cited by 35 publications

(53 citation statements)

References 26 publications

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“…Therefore, even though they are thermodynamically equivalent, ATP versus GTP utilization is biochemically segregated. It was therefore surprising that our research, as well as several literature reports using purified APH and MPH enzymes, revealed that many of these enzymes can use both ATP and GTP (2,13,16). Indeed, this preference was recently used as the basis for a proposed new nomenclature for APH(2Љ) enzymes (21).…”

Section: Discussionmentioning

confidence: 79%

“…These antibiotic kinases have largely been seen to use the phosphoryl donor ATP as the second substrate for antibiotic modification, consistent with the primacy of this nucleotide triphosphate (NTP) in primary metabolism and the intracellular concentration of ϳ3 mM in bacterial cells (1). During our efforts to characterize the structure and mechanism of macrolide antibiotic kinases, we were struck by the poor activity with ATP compared to GTP as a phosphoryl donor substrate in in vitro assays, exhibiting a selectivity that had been noted previously but was unexplored (13). This preference was surprising to us, given the reported intracellular concentrations of ATP versus GTP, which in bacterial and mammalian cells are reported to be on the order of 3-to 4-fold in excess of ATP (3; reviewed in reference 20).…”

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confidence: 99%

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Nucleotide Selectivity of Antibiotic Kinases

Shakya

Wright

2010

Antimicrob Agents Chemother

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Antibiotic kinases, which include aminoglycoside and macrolide phosphotransferases (APHs and MPHs), pose a serious threat to currently used antimicrobial therapies. These enzymes show structural and functional homology with Ser/Thr/Tyr kinases, which is suggestive of a common ancestor. Surprisingly, recent in vitro studies using purified antibiotic kinase enzymes have revealed that a number are able to utilize GTP as the antibiotic phospho donor, either preferentially or exclusively compared to ATP, the canonical phosphate donor in most biochemical reactions. To further explore this phenomenon, we examined three enzymes, APH(3)-IIIa, APH(2؆)-Ib, and MPH(2)-I, using a competitive assay that mimics in vivo nucleotide triphosphate (NTP) concentrations and usage by each enzyme. Downstream analysis of reaction products by high-performance liquid chromatography enabled the determination of partitioning of phosphate flux from NTP donors to antibiotics. Using this ratio along with support from kinetic analysis and inhibitor studies, we find that under physiologic concentrations of NTPs, APH(3)-IIIa exclusively uses ATP, MPH(2)-I exclusively uses GTP, and APH(2؆)-Ib is able to use both species with a preference for GTP. These differences reveal likely different pathways in antibiotic resistance enzyme evolution and can be exploited in selective inhibitor design to counteract resistance.Antibiotic modification is a major mechanism of resistance that impacts the efficacy of numerous antimicrobial drug classes. The enzymes that catalyze these group transfer mechanisms have evolved from precursor genes that encode proteins used to accomplish numerous metabolic tasks no doubt unrelated to drug resistance. Borrowing nomenclature from the oncogene field, we term such elements protoresistance genes. Biochemical and structural evidence has shown that protoresistance genes can be found in a number of key metabolic pathways, including cell wall biosynthesis and signal transduction (23). With respect to group transfer antibiotic resistance enzymes, these protoresistance genes encode protein and other small-molecule kinases, acetyltransferases, adenylyltransferases, ADP-ribosyltransferases, and glycosyltransferases. The addition of phosphoryl, acyl, adenyl, ribosyl, and glycosyl groups to the antibiotic scaffold alters the interaction with the cellular target to such a degree that the resistance phenotype results (6).The unifying biochemical logic of group transfer in antibiotic resistance is the co-opting of the normal cellular function of the protoresistance element by natural selection to include the modification of antibiotic molecules. Because group transfer enzymes require a second substrate (e.g., ATP, acetyl coenzyme A, or thymidine diphosphate glucose), these should retain the original specificity of the protoresistance enzyme since natural selection would not normally be expected to act on this site during resistance gene evolution.Antibiotic kinases represent a large superfamily of enzymes that covalently modify antibiotic...

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Section: Discussionmentioning

confidence: 79%

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confidence: 99%

Nucleotide Selectivity of Antibiotic Kinases

Shakya

Wright

2010

Antimicrob Agents Chemother

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“…The corresponding genes, which encode type I (mphA) and type II (mphB) macrolide 2Ј-phosphotransferases, confer a high level of resistance to several macrolides in E. coli. This resistance mechanism is a consequence of a modification of the active macrolide by phosphorylation within the sugar moiety (28,41). From the observed similarities, we can postulate that the ORF2 product might well be involved in phosphorylation of some intermediate substrate of the streptothricin biosynthetic pathway.…”

Section: Resultsmentioning

confidence: 93%

Streptothricin biosynthesis is catalyzed by enzymes related to nonribosomal peptide bond formation

1997

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In a search for strains producing biocides with a wide spectrum of activity, a new strain was isolated. This strain was taxonomically characterized as Streptomyces rochei F20, and the chemical structure of the bioactive product extracted from its fermentation broth was determined to be a mixture of streptothricins. From a genomic library of the producer strain prepared in the heterologous host Streptomyces lividans, a 7.2-kb DNA fragment which conferred resistance to the antibiotic was isolated. DNA sequencing of 5.2 kb from the cloned fragment revealed five open reading frames (ORFs) such that ORF1, -2, -3, and -4 were transcribed in the same direction while ORF5 was convergently arranged. The deduced product of ORF1 strongly resembled those of genes involved in peptide formation by a nonribosomal mechanism; the ORF2 product strongly resembled that of mphA and mphB isolated from Escherichia coli, which determines resistance to several macrolides by a macrolide 2-phosphotransferase activity; the ORF3 product had similarities with several hydrolases; and the ORF5 product strongly resembled streptothricin acetyltransferases from different gram-positive and gramnegative bacteria. ORF5 was shown to be responsible for acetyl coenzyme A-dependent streptothricin acetylation. No similarities in the databases for the ORF4 product were found. Unlike other peptide synthases, that for streptothricin biosynthesis was arranged as a multienzymatic system rather than a multifunctional protein.Insertional inactivation of ORF1 and ORF2 (and to a lesser degree, of ORF3) abolishes antibiotic biosynthesis, suggesting their involvement in the streptothricin biosynthetic pathway.During a screening process, a streptomycete producer of broad-range antibiotic activity was isolated from soil samples; this strain has been taxonomically characterized as Streptomyces rochei F20, and its active compound has been described as a mixture of streptothricins (mostly F and traces of D and E) (41a). The present paper describes the isolation, DNA sequence, and partial characterization of the biosynthetic pathway for streptothricin produced by this newly isolated strain. Streptothricin was one of the first actinomycete antibiotics to be described (56). It has a broad spectrum, with antibacterial as well as antifungal activity. Its chemical elucidation was described by Kusumoto and coworkers (30) (Fig. 1). It contains a heterocyclic ␤-amino acid (streptolidine), an amino sugar (4-carbamido-D-gulosamine), and, by amide linkage at C-2, a ␤-lysine chain which varies from one to six units in streptothricins F to A, respectively, and which includes seven units for streptothricin X (22, 31). More recently, other members of the family, which are chemically closely related to streptothricin, have been described (1,23,25).Members of the streptothricin family have, in addition to a potent inhibitory activity for prokaryotic protein synthesis (11), cytotoxicity which prevents their clinical or veterinary use. However, this may well be useful in situations in wh...

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“…However, macrolideinactivating enzymes, namely, EM esterases (1,20) and macrolide 2Ј-phosphotransferases (10,19), that mediate such resistance have been found in clinical isolates of Escherichia coli, which is naturally resistant to macrolides. Furthermore, almost all macrolide-inactivating enzymes are constitutively produced in E. coli (1,2,10,20). However, the production of macrolide 2Ј-phosphotransferase I [Mph(A); formerly MPH(2Ј)I] (22), which is a strong inactivator of 14-member ring macrolides, such as EM and oleandomycin (OL), is induced by EM in the original strain E. coli Tf481A (19).…”

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confidence: 99%