The Active Conformation of Avilamycin A Is Conferred by AviX12, a Radical AdoMet Enzyme*

The antibiotic avilamycin A is produced by Streptomyces viridochromogenes Tü57. Avilamycin belongs to the family of orthosomycins with a linear heptasaccharide chain linked to a terminal dichloroisoeverninic acid as aglycone. The gene cluster for avilamycin biosynthesis contains 54 open reading frames. Inactivation of one of these genes, namely aviX12, led to the formation of a novel avilamycin derivative named gavibamycin N1. The structure of the new metabolite was confirmed by mass spectrometry (MS) and NMR analysis. It harbors glucose as a component of the heptasaccharide chain instead of a mannose moiety in avilamycin A. Antibacterial activity tests against a spectrum of Gram-positive organisms showed that the new derivative possesses drastically decreased biological activity in comparison to avilamycin A. Thus, AviX12 seems to be implicated in converting avilamycin to its bioactive conformation by catalyzing an unusual epimerization reaction. Sequence comparisons grouped AviX12 in the radical S-adenosylmethionine protein family. AviX12 engineered with a His tag was overexpressed in Escherichia coli and purified by affinity chromatography. The iron sulfur cluster [Fe-S] present in radical AdoMet enzymes was detected in purified AviX12 by means of electron paramagnetic resonance spectroscopy.

Due to side effects and its poor water solubility, further development was stopped in 2000 (2). Both avilamycin and evernimicin were shown to inhibit protein biosynthesis by binding to the 50 S ribosomal subunit of the bacterial ribosomes (4 -6). Recently, we reported that methylation of G2535 and U2479 in domain V of the 23 S rRNA confers resistance to avilamycin by preventing the antibiotic from binding to the ribosome (7). This was in accordance with results obtained by footprinting avilamycin on Escherichia coli ribosomal subunits (8). Based on these data, it is suggested that avilamycin interacts with the ribosomal A site and interferes with initiation factor IF2 and tRNA binding.
The complete avilamycin biosynthetic gene cluster containing 54 open reading frames was sequenced (9). The corresponding genes were named avi. Based upon sequence similarities of the deduced proteins to enzymes of known function in the data base, a putative biosynthetic pathway for avilamycin has been proposed (9). Gene disruption experiments with putative methyltransferase genes have led to new avilamycin derivatives with enhanced water solubility, named gavibamycins (2). After deletion of aviB1 and aviO2, components of an incomplete pyruvate-dehydrogenase complex, an avilamycin derivative was obtained lacking the terminal acetyl residue at position C-4 of the eurekanate moiety of avilamycin A (10).
It is most likely that the biosynthesis of avilamycin starts with the formation of the unusual pentose L-lyxose. The next plausible steps toward formation of the heptasaccharide chain might be the unusual C1-C1 linkage between lyxose and mannose and subsequently the attachment of the eurekanate to the L-lyxose moiety. A knock-out mutation in aviE2, the gene encoding a UDP-glucuronic acid decarboxylase involved in the biosynthesis of lyxose from glucose, led to the breakdown of avilamycin A biosynthesis, confirming the presumption of the start of avilamycin biosynthesis in the coupling of lyxose and mannose (11). Inactivation of aviGT4, a putative glycosyltransferase, led to the formation of a new avilamycin derivative lacking the terminal eurekanate residue (11).
There still are genes with unknown function in the avilamycin biosynthetic gene cluster. One of these genes is aviX12, positioned in the center of the avilamycin biosynthetic gene cluster in proximity to methyltransferase and sugar biosynthetic genes (9). AviX12 shows no significant similarities to proteins of known function, but it contains a sequence motif typical for the radical S-adenosylmethionine (AdoMet) 2 protein family. Members of this family are among others involved in oxidative processes and making AviX12 a good candidate for oxidative reactions in the avilamycin biosynthesis, such as the building of the methylene bridge at the terminal eurekanate moiety or the orthoester linkages. To gain insight into the function of AviX12, aviX12 was inactivated, and the structure of a new avilamycin derivative that accumulated in this mutant was determined. Our results indicated the involvement of AviX12 in the formation of the biologically active conformation of avilamycin A by catalyzing an unusual epimerization reaction. Furthermore, aviX12 was modified with a His tag and overexpressed in E. coli. The protein was purified by affinity chromatography, and the bound [Fe-S] cluster was characterized by means of EPR spectroscopy.
General Genetic Manipulation, PCR, and Sequence Analysis-Routine methods were performed as described previously (16). Isolation of E. coli plasmid DNA, DNA restriction, DNA modification, and Southern hybridization were performed following the manufacturer's directions (Amersham Biosciences, Roche Diagnostics, Promega, and Stratagene). Streptomyces protoplast formation, transformation, and protoplast regeneration were performed as described previously (17). PCR was carried out using a GeneAmp PCR System 9700 (Applied Biosystems). Oligonucleotide primers were purchased at Qiagen-Operon GmbH. Computer-aided sequence analysis was done with the DNAsis software package (version 2.1, 1995; Hitachi Software Engineering). Data base searches were performed with the BLAST 2.0 program (18) on the server of the National Center for Biotechnology Information, Bethesda, MD.
Gene Inactivation of aviX12-AviX12 was PCR-amplified using oligonucleotides 5Ј-CTACCTGGAATTCCTGCTGACC-3Ј (aviX12F) and 5Ј-CAGCGCCGTCTAGAATCCGTAG-3Ј (aviX12R) containing engineered EcoRI and XbaI restriction sites (underlined), respectively. The resulting fragment was isolated with the Nucleospin Extract kit (Macherey Nagel), digested with EcoRI and XbaI, and ligated into pUC18 previously digested with the same enzymes, generating plasmid pUC-aviX12. The product was confirmed by DNA sequencing (4base lab GmbH). A unique SacII restriction site was altered by SacII restriction, subsequent treatment with T4 DNA polymerase, and religation. DNA sequencing confirmed the expected 2-bp deletion in aviX12, leading to frameshift mutation. After restriction with EcoRI and XbaI, the insert was transferred to plasmid pSP1 to generate IKX12. The inactivation construct was used to transform protoplasts of S. viridochromogenes GW4. Transformants were propagated in HA medium without erythromycin selection for 16 generations. Selection of erythromycinsensitive colonies gave the double crossover mutant S. viridochromoge-nes GW4-X12. The deletion within the gene was confirmed by PCR. PCR fragments obtained from mutant S. viridochromogenes GW4-X12 using primers 5Ј-GCGCGAGCCGGAGAAGCCGGAGA-3Ј (aviX12P-F) and 5Ј-TCTTGCGCAGCCCCTCGGCAACCA-3Ј (aviX12P-R) could not be cleaved by SacII (Fig. 2a), whereas the PCR fragment obtained from S. viridochromogenes GW4 could be digested by SacII.
Southern Hybridization-For Southern hybridization, genomic DNA from mutant and wild-type strains was completely digested with SacII, fractionated by agarose gel electrophoresis, and transferred to a positively charged nylon membrane (Hybond N ϩ ; Amersham Biosciences). The probe was a 1302-bp EcoRI/XbaI fragment from IKX12 labeled with digoxigenin-dUTP by the random priming method. Hybridization of the probe with DNA fragments on the nylon membrane was detected by the chromogenic method using procedures described by Roche Diagnostics.
Complementation of Mutant S. viridochromogenes GW4-X12-To determine clearly that the mutation event affected only the desired gene and not other genes, aviX12 was ligated behind the ermE* promoter of pSET-1cerm, and the resulting complementation construct pSETerm-aviX12 was introduced by protoplast transformation into the ⌬aviX12 mutant. The complementation led to restored gavibamycin production.
Expression and Purification of AviX12 (N-terminal His 6 -tagged)-For purification of expressed protein, E. coli strain BL21 (DE3)pLysS cells (Stratagene), carrying either pRSET-X12 with aviX12 or the pRSETb vector alone, were grown in LB broth containing 50 g/ml carbenicillin and 30 g/ml chloramphenicol to an A 600 ϭ 0.6. Protein expression was induced by the addition of 1 mM isopropyl-␤-D-thiogalactopyranoside, and growth was continued at 37°C for 4 h. Cells were harvested by centrifugation and stored at Ϫ20°C. The cell pellet from the 100-ml culture was resuspended in 4 ml of lysis buffer consisting of 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, and 5 mM dithiothreitol (DTT). The cells were broken by a single pass through a French pressure cell (Thermo Spectronic) at 700 pounds/square inch. After centrifugation, the supernatant fraction was used for the purification procedure. Protein was bound to nickel nitrilotriacetic acid-agarose (Qiagen) and loaded onto a column. The column was washed twice with washing buffer containing 50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM imidazole, and 5 mM DTT. The protein was recovered with elution buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole, and 5 mM DTT). The imidazole was removed by passing the protein over a PD-10 (Sephadex TM G-25; Amersham Biosciences) desalting column equilibrated in 10 mM Tris, pH 8.0, and 5 mM DTT. Expression and purification of AviX12 was monitored by SDS-PAGE (4% stacking gel and 12% resolving gel) followed by Coomassie Blue staining. The concentration of purified protein was estimated by the Bradford dye binding method (19). The molecular mass of the purified protein was determined by means of liquid chromatography/electrospray ionization-MS.
Isolation of Avilamycin Derivatives-Strains were grown in SG medium containing 2% glucose, 1% soy peptone, 0.1% CaCO 3 , 20 mM L-valine, and 1 ml of 0.1% CoCl 2 solution for 72 h at 28°C. Cultures were filtered, and the filtrate was extracted twice by ethyl acetate and evapo-rated to dryness. The mycelium was broken with acetone and filtered again. After evaporation of the acetone, the mycelium was also extracted with ethyl acetate and evaporated to dryness. Crude extracts from the filtrate and mycelium were combined and applied to a solid-phase extraction cartridge (SepPak C18; Waters Associates). The cartridge was eluted with 50 and 80% methanol. The 80% fraction contained ϳ90% of the avilamycin derivatives. The evaporated and lyophilized fractions were redissolved in acetonitrile and water. Further isolation was performed on an Agilent 1100 system using a semipreparative column (Zorbax SB-C18; 5 m, 9.4 m ϫ 150 mm). For elution, the following gradient profile was used: solvent A, 5 mM ammonium acetate in water; solvent B, acetonitrile, nonlinear gradient, 30 -50% of B within 20 min at a flow rate of 3.5 ml/min. A mass-based fraction collector was used to isolate the avilamycin derivatives. The final isolation step was performed using a gel permeation column (Plgel, 5 m; 100 Å, 300 ϫ 7.5 m; molecular weight Ͻ 4000). As the solvent, acetonitrile at a flow rate of 1 ml/min was used. Again, a mass-based fraction collector was used to isolate the avilamycin derivatives.
Biological Properties-The antimicrobial activity of the new derivative was determined by agar plate diffusion assay using Bacillus subtilis as the test strain. Susceptibility of staphylococci, streptococci, and enterococci to different derivatives was determined by the microdilution test according to National Committee for Clinical Laboratory Standards guidelines. Vancomycin was used as a standard to ensure the reliability of the determined minimum inhibitory concentrations.
Structural Characterization of the New Gavibamycin Derivative Accumulated by S. viridochromogenes GW4-X12-High performance liquid chromatography (HPLC)/electrospray ionization-MS was performed on an Agilent 1100 Series system with an electrospray chamber and a quadrupole detector. HPLC analysis was carried out on a Zorbax SB-C18 column (5 m, 4.6 ϫ 150 mm; Agilent) with a Zorbax SB-C18 precolumn (5 m, 4.6 ϫ 12.5 mm; Agilent). A nonlinear gradient from 20 to 70% acetonitrile in 0.5% acetic acid over 22 min at a flow rate of 0.5 ml/min was used. The column temperature was 23°C, and the UV detection wavelengths were 254 and 300 nm. The chamber settings were drying gas flow, 12 liters/min; nebulization pressure, 50 pounds/ square inch on the gauge; and drying gas temperature, 300°C. The samples were analyzed in positive and negative scan mode with a mass range of 700 -1500 Da. NMR analysis spectra were recorded on samples dissolved in 330 l of Me 2 SO-d 6 in SHIGEMI NMR (Tokyo, Japan) tubes at a temperature of 295 K on a Bruker DMX 750-MHz NMR spectrometer of the Bavaria NMR Center in Garching, Germany. The following spectra have been used for the structure elucidation: 13 C-1D, 13C-filtered nuclear Overhauser enhancement spectroscopy (20), correlated spectroscopy (21), heteronuclear single quantum coherence (22), and heteronuclear multiple bond coherence (23). The spectra were analyzed using XWINNMR version 3.6 (Bruker) and SPARKY version 3. 3 Spectroscopic Procedures-For UV-visible spectroscopy, A TIDAS-UVI/1001-1 diode array spectrometer with 512 diodes (J & M Analytische Mess-. Regeltechnik, EmbH, Aalen, Germany) was used for UVvisible spectroscopy. Spectra were recorded in the range from 200 to 600 nm in a buffer of 10 mM Tris, pH 8.0, and 5 mM DTT. The spectrum of a 1-ml aliquot of the preparation, as isolated, corresponded to the spectrum of the oxidized protein. The sample was mixed in a stirred optical cell with a few grains of sodium dithionite at room temperature while spectra were continuously recorded every 2 s. When there was no more spectral shift, the spectrum corresponded to the spectrum of the reduced protein. Spectra were processed using the Spectrachrom soft-ware package (J & M, Aalen, Germany). For EPR spectroscopy, the EPR spectra of the air-oxidized and dithionite-reduced preparation were recorded with an X-band spectrometer EMX 6/1 (Bruker) equipped with a helium flow cryostat (Oxford). Spectra were recorded at a 9.46-GHz microwave frequency, a modulation amplitude of 0.6 mT, time constant of 0.164 s, and scan rate of 17.9 mT/min. The optimal signal was measured at a temperature of 10 K and 1 milliwatt of microwave power. Computer simulations of the spectra were performed using the program SimFonia (Bruker), assuming no hyperfine interaction and gaussian line shape.
Calculation of the Avilamycin Conformation-Possible conformers of avilamycin were identified in a two-step molecular modeling process. A low energy conformation was constructed and optimized within the ArgusLab program package (25, 26) using a classical unified force field (27). In the relaxed molecule (by visual inspection), five C-O single bonds were identified that permit rotations of major fragments of the molecule around in steps of 120 degrees. The corresponding 3 5 ϭ 743 rotamers have been generated and checked for a significant atomic overlap using the repulsive part of the Lennard-Jones potential applying standard van der Waals radii. The geometry of each of the conformers has been optimized by a combination of Broyden-Fletcher-Goldfarb-Shanno and steepest descent techniques. The energy at the nine conformational minima has again been calculated using the unified force field referenced above.
GenBank TM Accession Number-The GenBank TM accession number for the DNA sequence reported in this paper is AAK83189.1.

RESULTS
Inactivation of aviX12-The inactivation of aviX12 was achieved by insertion of a frameshift mutation at a singular SacII restriction site central in aviX12 (Fig. 2a). As the wild-type strain for the inactivation experiment, S. viridochromogenes GW4 was chosen, a mutant carrying a deletion in the methyltransferase gene aviG4 leading to the production of gavibamycin A1 and A3 (Fig. 1). Protoplasts of S. viridochromogenes GW4 transformed with inactivation construct IKX12 were screened for a erythromycin-resistant phenotype. The strains selected were propagated without erythromycin selection. Two erythromycin-sensitive strains were examined by PCR; one of these proved the deletion of aviX12. This was verified by Southern hybridization (Fig. 2b). The mutant strain was designated S. viridochromogenes GW4-X12. HPLC analysis showed the production of new avilamycin derivatives in comparison to the wild-type strain.
Identification of New Avilamycin Derivatives in S. viridochromogenes GW4-X12-For analysis of secondary metabolite formation, both wild type and the ⌬aviX12 mutant were cultivated in production medium as described under "Experimental Procedures." Ethyl acetate extracts of the culture supernatants were analyzed by HPLC-UV and HPLC/electrospray ionization-MS. Gavibamycin A1 (1388 atomic mass units) and gavibamycin A3 (1390 u) were detected in extract of S. viridochromogenes GW4. In contrast, the ⌬aviX12 mutant accumulated four new compounds with atomic mass units of 1376, 1374 (main compound), 1332, and 1262, respectively. The mass of the main compounds indicate the loss of a methyl group in comparison to the main compounds of the wild-type gavibamycin A1 (1388 atomic mass units) and gavibamycin A3 (1390 atomic mass units). The new compounds were named gavibamycin N1 (1374 atomic mass units) and gavibamycin N3 (1376 atomic mass units). The other two compounds could be related to a derivative missing in addition to the methyl group, the acetate moiety at the eurekanate (1332 atomic mass units), and a derivative missing additionally the isobutyryl group at the lyxose moiety (1262 atomic mass units), respectively.
Structure Elucidation of the Gavibamycin A1 and A3 Derivatives-To elucidate the structure of the main compound of mutant S. viridochromogenes GW4-X12, this product was isolated as described under "Experimental Procedures." The NMR analysis of the mutants in this study is based on the completed 1 H and 13 C resonance assignment of avilamycin A and avilamycin C (2). The samples under investigation were prepared by feeding the bacteria 13 C-labeled L-methionine resulting in a partial 13 C-labeling of the products. For gavibamycin N1, carbons C-27, C-34, C-43, and C-61 showed enhanced signal intensities because of the labeling (the nomenclature is given in Fig. 1). The unambiguous identification of these carbons was done by the analysis of the 13 C-filtered nuclear Overhauser enhancement spectroscopy and the heteronuclear multiple bond coherence spectra. In Fig. 3, the resonances of atoms in the vicinity of C-37 (C-2 position in ring F) are shown in a section of the heteronuclear single quantum coherence spectra of gavibamycin N1 and avilamycin A. In comparison to avilamycin A, two methyl groups (C-7 and C-41) are missing in gavibamycin N1. Surprisingly, this is the same labeling pattern found in gavibamycin I1 (2). Because of the fact that it is very unlikely for an organism to have two enzymes with the same function, a further structural analysis was car-  ried out. Careful HPLC-UV analysis revealed that both compounds showed different retention times under the conditions described under "Experimental Procedures." As a first step, all 1 H and 13 C chemical shifts of gavibamycins I1 and N1 were compared. No significant difference was observed, except for the atoms in the vicinity of C-37. In a second step, the 3 J H,H couplings, which are directly linked to the dihedral angle of two hydrogens by the Karplus equation, were analyzed at the site of C-37. For the pair H-36/H-37 (the hydrogens bound to carbons C-36 and C-37, respectively) a 3 J coupling of 8 Ϯ 0.5 Hz was found for gavibamycin N1. This value is characteristic for an axial/axial orientation of H-36 and H-37 with respect to the ring. In contrast, for avilamycin A, a value of 3.5 Ϯ 0.5 Hz was found for the 3 J H-36,H-37 coupling, corresponding to an axial/equatorial orientation. This result indicated that a glucose is incorporated in gavibamycin N1 instead of a mannose in wildtype gavibamycin A1. Similar studies were carried out on the X12 mutation derivative with a molecular weight of 1332 atomic mass units. The conformation of H-36/H-37 was found to be axial/axial as for gavibamycin N1. Compared with gavibamycin N1 and avilamycin A, this compound is lacking the acetyl moiety in ring H, which is replaced by a hydrogen. This result is analogous to the changes reported for the mutations ITB1 and ITO2 (10).
Antibacterial Activity of Gavibamycin A1 and Gavibamycin N1-Extracts of wild type and S. viridochromogenes GW4-X12 were pretested against B. subtilis using the agar diffusion test. The extract of S. viridochromogenes GW4-X12 still showed antibiotic activity, but in comparison to the wild-type extract, it was lower. Furthermore, the gavibamycin N1 of S. viridochromogenes GW4-X12 and gavibamycin A1 of the wild-type strain were tested against a panel of pathogenic Gram-positive organisms, including two vancomycin-resistant strains, using the microdilution assay. The antibacterial activity of each derivative against the clinical isolates is presented in Table 1. All isolates were susceptible to gavibamycin A1 (minimum inhibitory concentration range Ͻ 0.5-8 g/ml). The changing of the mannose against a glucose in gavibamycin N1 drastically affected the activity against all of the tested organisms.
The Conformation of Avilamycin A-Of the nine conformers computed (see "Calculation of the Avilamycin Conformation"), seven lie within an energy interval of 50 kJ/mol around the absolute minimum. They either correspond to a linear shape of the molecule (two conformers) or create the general impression of a U shape (five conformers including the two lowest in energy). A typical example for the latter is shown in Fig. 4. Although the number of possible rotamers had been reduced considerably by the application of a molecular modeling procedure, only its combination with other criteria, such as the chemical accessibility of functional groups, could help to identify a unique, biologically active conformation.
Sequence Analysis of AviX12-The amino acid sequence of AviX12 comprises 396 amino acids. When we first published the sequence, BLAST searches did not reveal meaningful similarities to proteins with known functions in the data base. Recently, Sofia et al. (28) have identified the radical AdoMet protein superfamily by bioinformatic techniques. Despite low overall sequence similarity, all radical AdoMet enzymes contain an unconventional [4Fe-4S] cluster coordinated by three closely spaced cysteine residues, creating the defining CXXX-CXXC motif of this family (28). AviX12 contains this characteristic cysteine motif in its amino acid sequence precisely from amino acids Cys-156 to Cys-163, indicating that AviX12 belongs to the radical AdoMet family. The fourth iron of the cluster is coordinated by AdoMet. The function of radical AdoMet enzymes is to generate catalytic 5Ј-deoxyadenosyl radicals.
Expression and Purification of the N-terminal-His 6 -tagged AviX12-To demonstrate that AviX12 contains an [Fe-S] cluster, we cloned aviX12 into the expression vector pRSETb behind the T7 RNA polymerase promoter to generate an N-terminal hexahistidine fusion protein.    The protein was overexpressed in E. coli BL21 (DE3)pLysS. A protein of the predicted molecular mass of AviX12 (48,995 Da) was observed by SDS-PAGE in extracts of isopropyl-␤-D-thiogalactopyranoside-induced cells. AviX12 was purified by nickel chelation chromatography. The preparation was desalted and subjected to liquid chromatography/ electrospray ionization-MS. The molecular mass of the preparation was determined to 49,011 Da. The difference of 16 atomic mass units might be due to oxidation of methionine. Spectroscopic Characterization of the Iron-Sulfur Cluster-The UVvisible spectrum of the AviX12 preparation, as isolated, showed (beside the peak of the aromatic amino acids at 280 nm) a broad structureless absorbance from 300 to 550 nm, indicating the presence of a non-protein cofactor (Fig. 5). This signal was bleached by the addition of dithionite leading to a broad negative peak ϳ450 nm in the reduced minusoxidized difference spectrum (Fig. 5). This is a typical spectral feature of protein-bound [Fe-S] clusters (29). EPR spectra were recorded to determine the type of [Fe-S] cluster present in the AviX12 preparation. No EPR signals were detectable, with the sample reduced by dithionite in the temperature range from 5 to 100 K. However, the oxidized protein showed a signal at temperatures below 20 K, which was clearly seen at 10 K (Fig. 5). The signal exhibited an axial symmetry with a slight rhombic distortion and is typical for an [3Fe/4S] cluster. It was simulated with the following parameters: g x ϭ 2.007, g y ϭ 2.018, and g z ϭ 2.025; L x ϭ 1.65 mT; L y ϭ 1.1 mT, and L z ϭ 1.4 mT (Fig. 5). From spin quantitations, the presence of approximately one [3Fe/4S] cluster per AviX12 was calculated. Preliminary attempts to reconstitute an [4Fe/4S] cluster in the preparation under anaerobic conditions and in the presence of AdoMet were not successful, so far.

DISCUSSION
The biosynthesis of saccharide containing polyketides usually starts with the formation of the polyketide moiety. The sugar is attached to the polyketide at a later stage. In contrast, avilamycin biosynthesis starts with the formation of a disaccharide, and the polyketide moiety is attached to the hexasaccharide at the very end of the biosynthetic route. D-Mannose was discussed to be one component of the disaccharide starter molecule (11). Our data now indicate that the disaccharide is synthesized from D-glucose instead of D-mannose and that AviX12 is involved in C-2 epimerization of the glucose moiety after the whole avilamycin molecule is generated.
The reactions of most epimerases take place at a chiral carbon adjacent to an activating moiety, such as a carbonyl group, and the catalysis typically involves a simple deprotonation/reprotonation mechanism. Here epimerization takes place at an unactivated center. Most probably AviX12 and AdoMet generate a 5Јdeoxyadenosyl radical, which abstracts a hydrogen atom a position C-2 of the mannose moiety to form a radical intermediate. This intermediate undergoes epimerization by an unknown mechanism.
The antibiotic activity of gavibamycin N1, the main product of S. viridochromogenes GW4-X12, is very low. This strongly reduced activity might be explained by a conformational change of the whole molecule leading to an inability to bind to the ribosome. AviX12 activity is believed to induce an important conformational change of the molecule, resulting in its active form.
The calculation of the lowest energetic conformation of avilamycin A suggests an U form of the whole molecule. This conformation might be stabilized by hydrogen bonds between evalose and mannose on one hand and evalose and fucose on the other hand, as these sugars are located in the region of the angle of the molecule.
The transition of the low active gavibamycin N1 to the active avila-mycin A is mediated by AviX12. AviX12 was identified as a member of the radical AdoMet family. The presence of an [Fe-S] cluster in the preparation of AviX12 being typical for radical AdoMet enzymes was proven by spectroscopic methods. The EPR spectra clearly show that AviX12 contains an [3Fe-4S] cluster that is stable during the isolation of the protein. Different types of [Fe-S] clusters have been described for radical AdoMet enzymes. It was unequivocally shown that a [4Fe-4S] cluster is always present in the active enzymes (30,31). However, only the lysine 2,3-aminomutase seems to contain this cluster type after isolation (3). The other members of the family of radical AdoMet enzymes have been isolated either as apoenzymes or containing [2Fe-2S] or [3Fe-4S] clusters or some combinations of these components. Some preparations were reported to contain a [4Fe-4S] cluster as a minor component. In most cases, the active [4Fe-4S] cluster is obtained by reducing the preparations under anaerobic conditions. Our preparation did not contain any detectable traces of an [4Fe-4S] cluster. Radical AdoMet enzymes share structural features important for building the active sites of the proteins, which mostly contain the [4Fe-4S] cluster, the AdoMet cofactor, and the substrate shielding the radical intermediates from the surrounding medium. So far, we have not been able to reconstitute the functional [4Fe-4S] cluster by the addition of iron, AdoMet, and the substrate or any combinations thereof under anaerobic conditions. This is in agreement with the fact that our preparation shows no enzymatic activity.
It remains an open question whether the purification by means of affinity chromatography irreversibly damaged the protein or whether another cellular factor not present in our assay is needed for reconstitution of the cluster and enzymatic activity. It would be very interesting to learn whether the large avilamycin molecule is located in the active site of AviX12 and whether the conformational switch of the molecule after epimerization also takes places in the active site of the protein. To our best knowledge, AviX12 is the first example of a protein belonging to the radical AdoMet family being involved in the epimerization of a sugar.