Gentamicin X2 Research Articles

Structural and Functional Basis of GenB2 Isomerase Activity from Gentamicin Biosynthesis.

Aminoglycosides are essential antibiotics used to treat severe infections caused mainly by Gram-negative bacteria. Gentamicin is an aminoglycoside and, despite its toxicity, is clinically used to treat several pulmonary and urinary infections. The commercial form of gentamicin is a mixture of five compounds with minor differences in the methylation of one of their aminosugars. In the case of two compounds, gentamicin C2 and C2a, the only difference is the stereochemistry of the methyl group attached to C-6'. GenB2 is the enzyme responsible for this epimerization and is one of the four PLP-dependent enzymes encoded by the gentamicin biosynthetic gene cluster. Herein, we have determined the structure of GenB2 in its holo form in complex with PMP and also in the ternary complex with gentamicin X2 and G418, two substrate analogues. Based on the structural analysis, we were able to identify the structural basis for the catalytic mechanism of this enzyme, which was also studied by site-directed mutagenesis. Unprecedently, GenB2 is a PLP-dependent enzyme from fold I, which is able to catalyze an epimerization but with a mechanism distinct from that of fold III PLP-dependent epimerases using a cysteine residue near the N-terminus. The substitution of this cysteine residue for serine or alanine completely abolished the epimerase function of the enzyme, confirming its involvement. This study not only contributes to the understanding of the enzymology of gentamicin biosynthesis but also provides valuable details for exploring the enzymatic production of new aminoglycoside derivatives.

Synthesis of Gentamicin Minor Components: Gentamicin B1 and Gentamicin X2.

The clinical aminoglycoside antibiotic gentamicin is a mixture of several difficult-to-separate major and minor components. The relative inaccessibility of the minor components in particular complicates efforts to separate antibacterial activity from nephro- and/or ototoxicity and to clarify the origin of the potentially therapeutically important read-through activity. With a view to facilitating such studies, the synthesis of a fully and selectively protected garamine-based acceptor has been developed from readily available sisomicin. Glycosylation of this acceptor with a 6-azido-6,7-dideoxy-d-glycero-d-glucoheptopyranosyl donor affords gentamicin B1 after deprotection, whereas employment of a 2-azido-2-deoxy-d-glucopyranosyl donor under N,N-dimethylformamide-directed glycosylation conditions affords gentamicin X2 after deprotection.

The minor gentamicin complex component, X2, is a potent premature stop codon readthrough molecule with therapeutic potential.

Nonsense mutations, resulting in a premature stop codon in the open reading frame of mRNAs are responsible for thousands of inherited diseases. Readthrough of premature stop codons by small molecule drugs has emerged as a promising therapeutic approach to treat disorders resulting from premature termination of translation. The aminoglycoside antibiotics are a class of molecule known to promote readthrough at premature termination codons. Gentamicin consists of a mixture of major and minor aminoglycoside components. Here, we investigated the readthrough activities of the individual components and show that each of the four major gentamicin complex components representing 92–99% of the complex each had similar potency and activity to that of the complex itself. In contrast, a minor component (gentamicin X2) was found to be the most potent and active readthrough component in the gentamicin complex. The known oto- and nephrotoxicity associated with aminoglycosides preclude long-term use as readthrough agents. Thus, we evaluated the components of the gentamicin complex as well as the so-called “designer” aminoglycoside, NB124, for in vitro and in vivo safety. In cells, we observed that gentamicin X2 had a safety/readthrough ratio (cytotoxicity/readthrough potency) superior to that of gentamicin, G418 or NB124. In rodents, we observed that gentamicin X2 showed a safety profile that was superior to G418 overall including reduced nephrotoxicity. These results support further investigation of gentamicin X2 as a therapeutic readthrough agent.

Reaction Catalyzed by GenK, a Cobalamin-Dependent Radical S-Adenosyl-l-methionine Methyltransferase in the Biosynthetic Pathway of Gentamicin, Proceeds with Retention of Configuration.

Many cobalamin (Cbl)-dependent radical S-adenosyl-l-methionine (SAM) methyltransferases have been identified through sequence alignment and/or genetic analysis; however, few have been studied in vitro. GenK is one such enzyme that catalyzes methylation of the 6'-carbon of gentamicin X2 (GenX2) to produce G418 during the biosynthesis of gentamicins. Reported herein, several alternative substrates and fluorinated substrate analogs were prepared to investigate the mechanism of methyl transfer from Cbl to the substrate as well as the substrate specificity of GenK. Experiments with deuterated substrates are also shown here to demonstrate that the 6'-pro-R-hydrogen atom of GenX2 is stereoselectively abstracted by the 5'-dAdo· radical and that methylation occurs with retention of configuration at C6'. Based on these observations, a model of GenK catalysis is proposed wherein free rotation of the radical-bearing carbon is prevented and the radical SAM machinery sits adjacent rather than opposite to the Me-Cbl cofactor with respect to the substrate in the enzyme active site.

Structural Basis of the Selectivity of GenN, an Aminoglycoside N-Methyltransferase Involved in Gentamicin Biosynthesis.

Gentamicins are heavily methylated, clinically valuable pseudotrisaccharide antibiotics produced by Micromonospora echinospora. GenN has been characterized as an S-adenosyl-l-methionine-dependent methyltransferase with low sequence similarity to other enzymes. It is responsible for the 3″-N-methylation of 3″-dehydro-3″-amino-gentamicin A2, an essential modification of ring III in the biosynthetic pathway to the gentamicin C complex. Purified recombinant GenN also efficiently catalyzes 3″-N-methylation of related aminoglycosides kanamycin B and tobramycin, which both contain an additional hydroxymethyl group at the C5″ position in ring III. We have obtained eight cocrystal structures of GenN, at a resolution of 2.2 Å or better, including the binary complex of GenN and S-adenosyl-l-homocysteine (SAH) and the ternary complexes of GenN, SAH, and several aminoglycosides. The GenN structure reveals several features not observed in any other N-methyltransferase that fit it for its role in gentamicin biosynthesis. These include a novel N-terminal domain that might be involved in protein:protein interaction with upstream enzymes of the gentamicin X2 biosynthesis and two long loops that are involved in aminoglycoside substrate recognition. In addition, the analysis of structures of GenN in complex with different ligands, supported by the results of active site mutagenesis, has allowed us to propose a catalytic mechanism and has revealed the structural basis for the surprising ability of native GenN to act on these alternative substrates.

Delineating the Biosynthesis of Gentamicin X2, the Common Precursor of the Gentamicin C Antibiotic Complex

SummaryGentamicin C complex is a mixture of aminoglycoside antibiotics used worldwide to treat severe Gram-negative bacterial infections. Despite its clinical importance, the enzymology of its biosynthetic pathway has remained obscure. We report here insights into the four enzyme-catalyzed steps that lead from the first-formed pseudotrisaccharide gentamicin A2 to gentamicin X2, the last common intermediate for all components of the C complex. We have used both targeted mutations of individual genes and reconstitution of portions of the pathway in vitro to show that the secondary alcohol function at C-3″ of A2 is first converted to an amine, catalyzed by the tandem operation of oxidoreductase GenD2 and transaminase GenS2. The amine is then specifically methylated by the S-adenosyl-l-methionine (SAM)-dependent N-methyltransferase GenN to form gentamicin A. Finally, C-methylation at C-4″ to form gentamicin X2 is catalyzed by the radical SAM-dependent and cobalamin-dependent enzyme GenD1.

Genetic engineering combined with random mutagenesis to enhance G418 production in Micromonospora echinospora.

G418, produced by fermentation of Micromonospora echinospora, is an aminoglycoside antibiotic commonly used in genetic selection and maintenance of eukaryotic cells. Besides G418, M. echinospora produces many G418 analogs. As a result, the G418 product always contains impurities such as gentamicin C1, C1a, C2, C2a, gentamicin A and gentamicin X2. These impurities are less potent but more toxic than G418, but the purification of G418 is difficult because it has similar properties to its impurities. G418 is an intermediate in the gentamicin biosynthesis pathway. From G418 the pathway proceeds via successive dehydrogenation and aminotransferation at the C-6' position to generate the gentamicin C complex, but genes responsible for these steps are still obscure. Through disruption of gacJ, which is deduced to encode a C-6' dehydrogenase, the biosynthetic impurities gentamicin C1, C1a, C2 and C2a were all removed, and G418 became the main product of the gacJ disruption strain. These results demonstrated that gacJ is in charge of conversion of the 6'-OH of G418 into 6'-NH2. Disruption of gacJ not only eliminates the impurities seen in the original strain but also improves G418 titers by 15-fold. G418 production was further improved by 26.6% through traditional random mutagenesis. Through the use of combined traditional and recombinant genetic techniques, we produced a strain from which most impurities were removed and G418 production was improved by 19 fold.

Specificity and Promiscuity at the Branch Point in Gentamicin Biosynthesis

SummaryGentamicin C complex is a mixture of aminoglycoside antibiotics used to treat severe Gram-negative bacterial infections. We report here key features of the late-stage biosynthesis of gentamicins. We show that the intermediate gentamicin X2, a known substrate for C-methylation at C-6′ to form G418 catalyzed by the radical SAM-dependent enzyme GenK, may instead undergo oxidation at C-6′ to form an aldehyde, catalyzed by the flavin-linked dehydrogenase GenQ. Surprisingly, GenQ acts in both branches of the pathway, likewise oxidizing G418 to an analogous ketone. Amination of these intermediates, catalyzed mainly by aminotransferase GenB1, produces the known intermediates JI-20A and JI-20B, respectively. Other pyridoxal phosphate-dependent enzymes (GenB3 and GenB4) act in enigmatic dehydroxylation steps that convert JI-20A and JI-20B into the gentamicin C complex or (GenB2) catalyze the epimerization of gentamicin C2a into gentamicin C2.

Gene inactivation study on gntK, a putative C-methyltransferase gene in gentamicin biosynthesis from Micromonospora echinospora

GntK harbors methyltransferase-related cobalamin-binding domain and radical S-adenosylmethionine domain. The gntK-inactivation mutant of Micromonospora echinospora accumulated higher levels of gentamicin Cla and lower levels of gentamicin C1 and C2 isomers compared to the wild-type strain. Based on these results, we propose that GntK is involved in C-methylation on C-6′ in gentamicin X2 but is dispensable in gentamicin biosynthesis.

Characterization of Saccharomyces cerevisiae mutants supersensitive to aminoglycoside antibiotics

We describe mutants of Saccharomyces cerevisiae that are more sensitive than the wild type to the aminoglycoside antibiotics G418, hygromycin B, destomycin A, and gentamicin X2. In addition, the mutants are sensitive to apramycin, kanamycin B, lividomycin A, neamine, neomycin, paromomycin, and tobramycin--antibiotics which do not inhibit wild-type strains. Mapping studies suggest that supersensitivity is caused by mutations in at least three genes, denoted AGS1, AGS2, and AGS3 (for aminoglycoside antibiotic sensitivity). Mutations in all three genes are required for highest antibiotic sensitivity; ags1 ags2 double mutants have intermediate antibiotic sensitivity. AGS1 was mapped 8 centimorgans distal from LEU2 on chromosome III. Analyses of yeast strains transformed with vectors carrying antibiotic resistance genes revealed that G418, gentamicin X2, kanamycin B, lividomycin A, neamine, and paromomycin are inactivated by the Tn903 phosphotransferase and that destomycin A is inactivated by the hygromycin B phosphotransferase. ags strains are improved host strains for vectors carrying the phosphotransferase genes because a wide spectrum of aminoglycoside antibiotics can be used to select for plasmid maintenance.

Sagamicin and the related aminoglycosides: fermentation and biosynthesis. I. Biosynthetic studies with the blocked mutants of Micromonospora sagamiensis.

The mutants blocked in the gentamicin C1 production were derived from a sagamicin producing strain of Micromonospora sagamiensis. The intermediates produced by these mutants were isolated and properly identified. Comparing the biotransformation activities in the resting cells of the mutants with those of a DOS idiotroph, KY 11525, the blocked steps in sagamicin and gentamicin biosynthesis were proposed in each mutant. Mutant KY 11564 was found to produce gentamicin C2a (C-6'-epimer of gentamicin C2) together with gentamicin C1a and sagamicin. KY 11525 transformed gentamicin C2a into C2 and C1, whereas KY 11564 lacked the activity. KY 11565 produced gentamicin X2 and antibiotic G-418, and lacked 6'-amino substitution activities. KY 11566 appeared to be partially blocked in 6'-N-methylation activities, and the major products were gentamicin C2a and C1a. From these results, sagamicin biosynthesis in M. sagamiensis is discussed.

Biosynthetic pathway leading to gentamicin C2b.

Incubation of Micromonospora purpurea SC 1210 (NRRL 5467) with L-[methyl-14C]methionine yielded [methyl-14C]gentamicin A and methyl-14C-labeled antibiotic JI-20A in a molar radioactivity ratio of 9:2. We did not isolate methyl-14C-labeled gentamicin X2, which was expected as an intermediate based on the biosynthetic pathways proposed by others. Addition of methyl-14C-labeled antibiotic JI-20A to M. purpurea SC 1124 (NRRL 8102) yielded [methyl-14C]-gentamicin C1a and [methyl-14C]gentamicin C2b in variable molar radioactivity ratios. These data do not support the biosynthetic pathway leading to gentamicin C2b proposed by Testa and Tilley.

Semisynthetic aminoglycoside antibacterials. Part II. Synthesis of gentamicin X2 and related compounds.

Gentamicin X2, naturally occurring aminoglycoside antibiotic produced as a minor component together with the clinically important gentamicin C complex by Micromonospora purpurea, has been synthesized by glycosylation of suitably protected garamine derivatives. The synthesis of the α-glycoside was accomplished by means of the Lemieux–Nagabhushan reaction as well as by using a Koenigs–Knorr reaction. The latter reaction was also used to prepare 4-O-β-analogue of gentamicin X2. The syntheses of other analogues of gentamicin X2, namely 4-O-[2-amino-2-deoxy-α-D-mannopyranosyl], 4-O-[2-amino-2-deoxy-α-D-galactopyranosyl], 4-O-[2-amino-2-deoxy-α- and -β-D-glucofuranosyl], 4′-O-[2-amino-2-deoxy-α-D-glucopyranosyl], 4-O-α-D-glucopyranosyl, 4-O-α-D-talopyranosyl, and 4-O-2-deoxy-α-D-galactopyranosyl derivatives of garamine, are also described.

Semisynthetic aminoglycoside antibacterials. Part III. Synthesis of analogues of gentamicin X2modified at the 3′-position

In order to prevent inactivation of gentamicin X2 by phosphorylation, a series of 3′-modified derivatives was prepared. These included 3′-deoxygentamicin X2, O-2-amino-2-deoxy-α-D-allopyranosyl-(1 → 4)-garamine, and 3′-O-methylgentamicin X2, all of which were synthesized by application of the Lemieux-Nagabhushan reaction, to a suitably protected garamine intermediate. The formation of O-2-amino-2-deoxy-3-O-methyl-β-D-manno-pyranosyl-(1 → 4)-garamine during the synthesis of 3′-O-methylgentamicin X2 by the Lemieux-Nagabhushan reaction is reported. The solution conformations of the novel 4-O-β-D-mannopyranosyl and 4-O-β-D-glucopyranosyl derivatives are discussed. The formation of nitroglucals during the preparation of the nitroso-chloro-adducts is described.