Intermediates of rifamycin polyketide synthase produced by an Amycolatopsis mediterranei mutant with inactivated rifF gene

Abstract

Rifamycin B biosynthesis in Amycolatopsis mediterranei N/813 was inactivated by introducing a small deletion in the rifF gene situated directly downstream of the rifamycin polyketide synthase (PKS) gene cluster. The corresponding mutant strain produced a series of linear intermediates of rifamycin B biosynthesis that are most probably generated by obstruction of the normal release of the end product of the rifamycin PKS. This result provides evidence that the rifF gene product catalyses the release of the completed linear polyketide from module 10 of the PKS and the intramolecular macrocyclic ring closure by formation of an amide bond, as indicated by sequence similarity of this protein to amide synthases. The chemical structures of the new rifamycin polyketide synthase intermediates released from modules 4 to 10 were determined by spectroscopic methods (UV, IR, NMR and MS) and gave insight into the reaction steps of rifamycin ansa chain biosynthesis and the timing of the formation of the naphthoquinone ring. The intermediates released from modules 6 and 8 were isolated as lactones formed by the terminal carboxyl group ; proton NMR double resonance and ROESY(rotated frame nuclear Overhauser enhancement spectroscopy) experiments enabled the deduction of the relative configurations in the linear chain which correspond to the known absolute stereochemistry of rifamycin B.Keywords : antibiotic biosynthesis, ansamycins, amide synthase, gene replacement, pathway engineering INTRODUCTIONRifamycins are clinically important ansamycin antibiotics, composed of a naphthalenic chromophore spanned by a long aliphatic ansa chain. The rifamycins and the semisynthetic drugs derived from them exert their antibiotic activity by specific inhibition of bacterial DNA-dependent RNA polymerase (Wehrli, 1977). At higher concentrations, these antibiotics also inhibit the RNA-dependent DNA polymerase of retroviruses (Szabo et al., 1976 bacterium leprae, causative agents of tuberculosis and leprosy, respectively, they are also active against a variety of other organisms, including bacteria and viruses (Szabo et al., 1976 ;Oppenheim et al., 1986 ;Barakett et al., 1993 ;Bachs et al., 1992). Furthermore, it was shown recently that rifampicin-containing regimens are able to cure staphylococcal implant-related infections (Zimmerli et al., 1998).The identification and sequencing of the rifamycin polyketide synthase (PKS) gene cluster by our group (Schupp et al., 1998) and by August et al. (1998) Our interest in further studying rifamycin B biosynthesis led us to undertake the inactivation of the rifF gene, situated directly downstram of the PKS genes, and to analyse the effect of this mutation on rifamycin biosynthesis. The rifF gene product has been characterized as rifamycin amide synthase by sequence homologies to different arylamine N-acetyltransferases (August et al., 1998) and the putative function of the RifF protein in rifamycin biosynthesis was suggested to be a cyclase, catalysing the formation of an intramolecular amide bond between ...

Identification of tailoring genes involved in the modification of the polyketide backbone of rifamycin B by Amycolatopsis mediterranei S699

“…However, mutant strain MT0031ABH, in which a 21 kb fragment of the post-PKS modification genes has been deleted, did not accumulate proansamycin X, but instead the tetraketides SY4b and desacetyl-SY4b were produced. These compounds have previously been isolated from knock-out mutants of the amide synthase RifF, as a result of premature release of the polyketide chain intermediates from the enzyme, starting from the tetraketide through the undecaketide (Stratmann et al, 1999;Yu et al, 1999). Structure identification of these intermediates showed that from the pentaketide onwards they contain a naphthalene ring, suggesting that the formation of the naphthalene moiety of rifamycin B takes place during the chain extension of the polyketide, specifically between the tetraketide and the pentaketide stage.…”

“…Based on results of the present study and others (Stratmann et al, 1999;Yu et al, 1999), it was clear that the core naphthalene ring moiety of rifamycin is constructed during the polyketide chain extension process. However, the fact that the reaction may require a catalytic enzyme, whose gene is located in the post-PKS modification region and functions in trans with the PKS, is rather unusual.…”

“…Instead, the mutant strain produced the tetraketide products SY4b and desacetyl-SY4b (the cyclic forms of P8/1-OG, Fig. 4B), which have previously been shown to be premature release products when the amide synthase (RifF) is inactivated (Stratmann et al, 1999;Yu et al, 1999). Negative-ion ESI-MS tandem analysis of desacetyl-SY4b gave fragment ions at m/z 246, 228,190,172,153,136, which are consistent with the expected fragmentation patterns for desacetyl-SY4b (Fig.…”

“…The amide synthase (RifF) catalyses the release of this linear product from the PKS complex and its cyclization via intramolecular amide formation. Inactivation of RifF results in the premature release of a series of polyketides of different chain length from the enzyme complex (Stratmann et al, 1999;Yu et al, 1999), and reveals that the formation of the naphthalene ring of rifamycin occurs during the chain extension. However, further studies are required to determine the mechanism and genes responsible for the construction of the naphthalene core motif.…”

The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum

“…The AT domain of module 3 of AsmB, which is predicted to recognize the ''glycolate'' extender unit, shows no unusual sequence signature that would distinguish it from other AT domains. Located immediately downstream of asmD is asm9, which encodes a protein with a high degree of similarity to RifF, the chain-terminating, cyclizing enzyme of rifamycin biosynthesis (33,34). It is proposed that the C terminus of the fully extended polyketide chain is transferred from AsmD to the conserved Cys-73 on the Asm9 protein, followed by intramolecular amide bond formation with the aromatic amino group to release a 19-membered macrocyclic lactam, proansamitocin (Fig.…”