Aclacinomycin 10-Hydroxylase Is a Novel Substrate-assisted Hydroxylase Requiring S-Adenosyl-l-methionine as Cofactor

Aclacinomycin 10-hydroxylase is a methyltransferase homologue that catalyzes a S-adenosyl-l-methionine (AdoMet)-dependent hydroxylation of the C-10 carbon atom of 15-demethoxy-ϵ-rhodomycin, a step in the biosynthesis of the polyketide antibiotic β-rhodomycin. S-Adenosyl-l-homocysteine is an inhibitor of the enzyme, whereas the AdoMet analogue sinefungin can act as cofactor, indicating that a positive charge is required for catalysis. 18O2 experiments show that the hydroxyl group is derived from molecular oxygen. The reaction further requires thiol reagents such as glutathione or dithiothreitol. Incubation of the enzyme with substrate in the absence of reductant leads to the accumulation of an intermediate with a molecular mass consistent with a perhydroxy compound. This intermediate is turned into product upon addition of glutathione. The crystal structure of an abortive enzyme-AdoMet product ternary complex reveals large conformational changes consisting of a domain rotation leading to active site closure upon binding of the anthracycline ligand. The data suggest a mechanism where decarboxylation of the substrate results in the formation of a carbanion intermediate, which is stabilized by resonance through the aromatic ring system of the anthracycline substrate. The delocalization of the electrons is facilitated by the positive charge of the cofactor AdoMet. The activation of oxygen and formation of a hydroxyperoxide intermediate occurs in a manner similar to that observed in flavoenzymes. Aclacinomycin-10-hydroxylase is the first example of a AdoMet-dependent hydroxylation reaction, a novel function for this cofactor. The enzyme lacks methyltransferase activity due to the positioning of the AdoMet methyl group unfavorable for a SN2-type methyl transfer to the substrate. Aclacinomycin 10-hydroxylase is a methyltransferase homologue that catalyzes a S-adenosyl-l-methionine (AdoMet)-dependent hydroxylation of the C-10 carbon atom of 15-demethoxy-ϵ-rhodomycin, a step in the biosynthesis of the polyketide antibiotic β-rhodomycin. S-Adenosyl-l-homocysteine is an inhibitor of the enzyme, whereas the AdoMet analogue sinefungin can act as cofactor, indicating that a positive charge is required for catalysis. 18O2 experiments show that the hydroxyl group is derived from molecular oxygen. The reaction further requires thiol reagents such as glutathione or dithiothreitol. Incubation of the enzyme with substrate in the absence of reductant leads to the accumulation of an intermediate with a molecular mass consistent with a perhydroxy compound. This intermediate is turned into product upon addition of glutathione. The crystal structure of an abortive enzyme-AdoMet product ternary complex reveals large conformational changes consisting of a domain rotation leading to active site closure upon binding of the anthracycline ligand. The data suggest a mechanism where decarboxylation of the substrate results in the formation of a carbanion intermediate, which is stabilized by resonance through the aromatic ring system of the anthracycline substrate. The delocalization of the electrons is facilitated by the positive charge of the cofactor AdoMet. The activation of oxygen and formation of a hydroxyperoxide intermediate occurs in a manner similar to that observed in flavoenzymes. Aclacinomycin-10-hydroxylase is the first example of a AdoMet-dependent hydroxylation reaction, a novel function for this cofactor. The enzyme lacks methyltransferase activity due to the positioning of the AdoMet methyl group unfavorable for a SN2-type methyl transfer to the substrate. Anthracyclines are a group of aromatic polyketides and their derivatives, some of which are in clinical use in cancer therapy. They act principally by inhibiting topoisomerase II (1Larsen A.K. Escargueil A.E. Skladanowski A. Pharmacol. Ther. 2003; 99: 167-181Crossref PubMed Scopus (356) Google Scholar). Complete or partial biosynthetic clusters for daunorubicin (2Strohl W.R. Dickens M.L. Rajgarhia V.B. Woo A.J. Priestley N.D. Strohl W.R. Biotechnology of Antibiotics. Marcel Decker, New York1997: 577-657Google Scholar), doxorubicin (3Otten S.L. Stutzman-Engwall K.J. Hutchinson C.R. J. Bacteriol. 1990; 172: 3427-3434Crossref PubMed Google Scholar), nogalamycin (4Torkkell S. Kunnari T. Palmu K. Mäntsälä P. Hakala J. Ylihonko K. Mol. Genet. Genomics. 2001; 266: 276-288Crossref PubMed Scopus (66) Google Scholar), aclacinomycin (5Fujii I. Ebizuka Y. Chem. Rev. 1997; 97: 2511-2523Crossref PubMed Scopus (42) Google Scholar, 6Räty K. Kantola J. Hautala A. Hakala J. Ylihonko K. Mäntsälä P. 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The success of combinatorial biosynthesis rests on the ability of individual enzymes to accept non-natural substrates. A final product can only be produced if each and every step in the pathway yields a product that can be used by the next enzyme in the chain. Therefore, mechanistic insights, in particular knowledge regarding the structural basis of substrate recognition and specificity, could possibly facilitate the use of this approach by the re-design of the specificity profile of the enzymes in these pathways. As part of our efforts to investigate the structural and mechanistic enzymology of the biosynthetic pathway(s) of anthracyclines, we have studied the enzyme aclacinomycin 10-hydroxylase (RdmB) 1The abbreviations used are: RdmB, aclacinomycin-10-hydroxylase; AknA, aclacinomycin A (aklavinone-rhodosamine-deoxyfucose-cinerulose A); RdmC, aclacinomycin-15-methylesterase; DnrK, carminomycin-O-methyltransferase; DbrA, 11-deoxy-β-rhodomycin A; DcmaA, 10-decarboxymethylaclacinomycin A; DmaA, 15-demethylaclacinomycin A; HPLC, high pressure liquid chromatography; DTT, dithiothreitol; M-ϵ-T, 4-O-methyl-ϵ-rhodomycin T (4-O-methyl-ϵ-rhodomycinone-rhodosamine); AdoHcy, S-adenosyl-l-homocysteine; AdoMet, S-adenosyl-l-methionine; LC, liquid chromatography; MS, mass spectrometry; MES, 4-morpholineethanesulfonic acid. (11Jansson A. Niemi J. Lindqvist Y. Mäntsälä P. Schneider G. J. Mol. Biol. 2003; 334: 269-280Crossref PubMed Scopus (33) Google Scholar). This enzyme participates in rhodomycin biosynthesis in Streptomyces purpurascens and is responsible for the insertion of the 10-hydroxyl substituent observed in β-rhodomycinone glycosides (Fig. 1). The amino acid sequence and the three-dimensional structure of this protein clearly suggested that it is a AdoMet-dependent methyltransferase. The closest known relative (52% amino acid sequence identity) is carminomycin-O-methyltransferase (DnrK) (12Jansson A. Koskiniemi H. Mäntsälä P. Niemi J. Schneider G. J. Biol. Chem. 2004; 279: 41149-41156Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), one of the first anthracycline biosynthetic enzymes to be studied in vitro (13Madduri K. Torti F. Colombo A.L. Hutchinson C.R. J. Bacteriol. 1993; 175: 3900-3904Crossref PubMed Google Scholar). Despite this close relationship to a methyltransferase, the occurrence of the 10-hydroxyl group in products of combinatorial biosynthesis was dependent on the presence of the rdmB gene (14Wang Y. Niemi J. Airas K. Ylihonko K. Hakala J. Mäntsälä P. Biochim. Biophys. Acta. 2000; 1480: 191-200Crossref PubMed Scopus (19) Google Scholar). Subsequently, it was shown (15Wang Y. Niemi J. Mäntsälä P. FEMS Microbiol. Lett. 2002; 208: 117-122Crossref PubMed Google Scholar) that aclacinomycin-15-methylesterase (RdmC) and RdmB acting in sequence in vitro produced the 10-hydroxyl group from the 10-carboxymethyl substituent observed in aclacinomycins. The reaction catalyzed by RdmB is particularly intriguing, because the enzyme does not use any of the cofactors usually associated with hydroxylases such as flavins and/or metal ions to activate molecular oxygen. It is also noteworthy that the enzyme lacks methyltransferase activity (7Niemi J. Mäntsälä P. J. Bacteriol. 1995; 177: 2942-2945Crossref PubMed Google Scholar, 14Wang Y. Niemi J. Airas K. Ylihonko K. Hakala J. Mäntsälä P. Biochim. Biophys. Acta. 2000; 1480: 191-200Crossref PubMed Scopus (19) Google Scholar, 15Wang Y. Niemi J. Mäntsälä P. FEMS Microbiol. Lett. 2002; 208: 117-122Crossref PubMed Google Scholar). A previous crystallographic study (11Jansson A. Niemi J. Lindqvist Y. Mäntsälä P. Schneider G. J. Mol. Biol. 2003; 334: 269-280Crossref PubMed Scopus (33) Google Scholar) had shown that RdmB has the typical fold of small molecule methyltransferases and that AdoMet is bound close to the putative active site. However, the absence of any bound anthracycline ligand in the active site prevented detailed mechanistic conclusions as to this enigmatic hydroxylation reaction. Here, we present evidence that RdmB is a hydroxylase with a novel mechanism. In particular, these studies suggest a new function for AdoMet in enzyme reactions. RdmB and its closest homologue, DnrK, are particularly striking and illustrative examples of how a novel function in a given metabolic pathway can develop by divergent evolution. Expression and Purification—Recombinant RdmB was produced and purified as described previously for DnrK (12Jansson A. Koskiniemi H. Mäntsälä P. Niemi J. Schneider G. J. Biol. Chem. 2004; 279: 41149-41156Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Selenomethionine-substituted RdmB was prepared as described (11Jansson A. Niemi J. Lindqvist Y. Mäntsälä P. Schneider G. J. Mol. Biol. 2003; 334: 269-280Crossref PubMed Scopus (33) Google Scholar). The purification of recombinant methylesterase RdmC followed procedures described elsewhere (14Wang Y. Niemi J. Airas K. Ylihonko K. Hakala J. Mäntsälä P. Biochim. Biophys. Acta. 2000; 1480: 191-200Crossref PubMed Scopus (19) Google Scholar). In Vitro Mutagenesis—RdmB was mutated using the QuikChange mutagenesis system (Stratagene). After sequencing, the mutant proteins were produced as described for the wild-type enzyme; however, due to solubility problems, the fermentation temperature was lowered to 25 °C for C165S. HPLC and LC-MS—HPLC of anthracyclines was performed on Li-Chrospher 100 RP18 columns with a mixture of 70:30 acetonitrile and 60 mmol/liter ammonium acetate buffer, pH 3.6, as eluent. For analytical HPLC, a 250 × 4-mm, 5-μm particle size column at a flow rate of 1 ml/min was coupled to a Shimadzu VP series chromatography system with a diode array detector, which was also used to record the UV-visible spectra of the products. In preparative HPLC, a 250 × 10-mm, 10-μm particle size column at a flow rate of 2.5 ml/min was used. HPLC of AdoMet and was as described previously P. S. J. 2001; PubMed Scopus Google Scholar). was performed with a system positive have observed that the substrate for RdmB, A is and in to 10-decarboxymethylaclacinomycin and (Fig. 1). Therefore, of enzyme were used. In a coupled the substrate was in by with purified RdmC in buffer, pH in the presence of 1 mmol/liter AdoMet, and the RdmB to be for 1 at The reaction was and the anthracycline products were by using columns to the The products were by analytical In the of were prepared as was in the presence of the methyltransferase RdmC and glutathione as described glycosides at pH and were by with of and purified was by as This was to a reaction mixture buffer, pH 1 mmol/liter and AdoMet with the RdmB to be with and product as in the coupled was by to a of mmol/liter to a coupled reaction that had to and the at and addition of 1 glutathione had A reaction mixture RdmB was used as and of a mixture were into in of a and substrate and for The were and or with as a or with the addition of 18O2 to for oxygen in the products. reaction was in the absence of glutathione using an enzyme prepared The products of these were as and the molecular of these were by mass and of RdmB in mmol/liter buffer, pH was with mmol/liter 11-deoxy-β-rhodomycin mmol/liter AdoMet, and mmol/liter was using of the were with of and to at and were 1 using a consisting of ammonium and buffer, pH data to a was at at 100 using to the The to group and a of The complex was in the manner as the enzyme A. Niemi J. Mäntsälä P. Schneider G. Biol. 2003; PubMed Scopus Google Scholar) with the addition of mmol/liter sinefungin of AdoMet to the The of group were in A data was at at the to a of The were in a at 100 using as a for of the data were and with the Biol. 1994; PubMed Scopus Google Scholar). of the Biol. 1994; PubMed Scopus Google Scholar) was used for of the was by the and of the in and of the with the G. J. Scopus Google Scholar). The for the data are given in of data and crystallographic of of of amino of from from of in in in in a new and structure of the complex was by molecular using the A.J. 2004; PubMed Scopus Google Scholar). A derived from the of the complex of RdmB with AdoMet consisting of the and a for this part of one in the This was and a rotation and was to the of the The J. 1997; Scopus Google Scholar) was used to the molecule in the which a of and of The structure of the ternary complex of product large and in the of the domain relative to each with the structure of the the complex of The by molecular was by and using the J. S. A. PubMed Scopus Google Scholar). The structure of the complex was also by molecular because the were to of the complex as by in and with and The G. Biol. 1997; PubMed Scopus Google Scholar) was for of the using the a of for every data was for After an of and were used. were to the data to The fold was whereas the of the structure were were by A. R. Nat. Biol. PubMed Scopus Google Scholar) and by the The of the was with J. 1993; Google Scholar) in I. and sequence were using D.J. J. Mol. Biol. 1990; PubMed Scopus Google Scholar) and 1994; PubMed Scopus Google Scholar), and structure were with G. J. Scopus Google Scholar) and the in using The were using The Scholar), A. K. J. Mol. Scopus Google Scholar), and A new HPLC was to the products and of the RdmB reaction. In particular, the use of with In mass of anthracycline by this we a to the The to the the mass spectra of and in the absence and presence of oxygen. In and and and were by not DmaA, produced from by RdmC, is and in (Fig. A and Therefore, it was or the was the of partial purification of as substrate of the RdmB reaction. As can be from this with the DcmaA, the product of the was purified by preparative HPLC and was not by RdmB (Fig. In previous (14Wang Y. Niemi J. Airas K. Ylihonko K. Hakala J. Mäntsälä P. Biochim. Biophys. Acta. 2000; 1480: 191-200Crossref PubMed Scopus (19) Google Scholar, 15Wang Y. Niemi J. Mäntsälä P. FEMS Microbiol. Lett. 2002; 208: 117-122Crossref PubMed Google Scholar), thiol reagents were to the reaction as the enzyme was prepared with 1 mmol/liter DTT, was a small of thiol However, we observed that a and consistent reaction was 1 mmol/liter or glutathione was the of the reaction, the of glutathione was with glutathione In a reaction with of the substrate AknA, were to a product and glutathione. enzyme was prepared the of thiol reagents and was to the reaction a new product was observed in addition to (Fig. This at in HPLC and had a mass consistent with a molecule an oxygen atom with the final (Fig. the UV-visible of was from that of The size of the from to glutathione was to the reaction, the upon further The of the reaction upon thiol reagents to investigate or not a of the enzyme be in the hydroxylation reaction. of the three-dimensional structure of RdmB suggested only one close to the active site. prepared mutant was active as the enzyme, that this is not for catalysis. amino which is in the active site and which RdmB and the active methyltransferase DnrK, is The of this amino acid by mutagenesis with a not the hydroxylase activity of the products were in the The hydroxylation reaction was dependent on oxygen. prepared in a in the absence of only the product product of the RdmC and the product were The addition of oxygen to the reaction mixture to the formation of the product In the where was by the molecular mass of by In the absence of glutathione and in the presence of the reaction in the formation of the product with the molecular mass by consistent with the of oxygen in this (Fig. The hydroxylation reaction in the as a for the reaction. The reaction was dependent on AdoMet and the addition of this cofactor the of product by to (Fig. The activity the addition of AdoMet is due to of AdoMet bound to the enzyme The addition of the reaction to with AdoMet of AdoMet and by HPLC from reaction not also that AdoMet is not in the reaction. The methyltransferase inhibitor sinefungin was to the hydroxylation reaction and was to for AdoMet (Fig. of RdmB of The domain is in an and is by and only The domain is small and The domain has an fold by a by on This domain the and the cofactor, AdoMet. The RdmB in the ternary complex in and and are in structure with the complex In the ternary complex (Fig. and of are because this of sequence has in the product the that are in the complex a in the ternary The is also in the ternary In this a of for and for is a large in RdmB upon binding of (Fig. of the and ternary using only the reveals a in the relative of the in the The conformational can be described as a rotation of the domain by an in a of this domain by As a of the domain and close the of the substrate and the and also to the bound product in the ternary and are close to the of the substrate in the whereas in the ternary and of are AdoMet AdoMet cofactor is bound in the in the structure as in the It the and with the (Fig. The only in the of is the absence of the the group of AdoMet and the hydroxyl group of in the ternary This is on a and to be in the structure of the binding site in RdmB is the and and from are in binding of the The binding is by that the aromatic ring (Fig. one from the participates in these The in these are through its carbon and The bound ligand only one to the enzyme from the oxygen in to the of The from the binding into the The of the are that are bound to the in sequence and three-dimensional structure to a of AdoMet-dependent of the closest is DnrK, which catalyzes at the hydroxyl group in the biosynthesis of the anthracycline daunorubicin in Streptomyces (13Madduri K. Torti F. Colombo A.L. Hutchinson C.R. J. Bacteriol. 1993; 175: 3900-3904Crossref PubMed Google Scholar). The three-dimensional structure of DnrK in complex with the products and was (12Jansson A. Koskiniemi H. Mäntsälä P. Niemi J. Schneider G. J. Biol. Chem. 2004; 279: 41149-41156Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). A of the ternary of the enzymes that are similar with a of for The only is the amino that is to the substrate binding in RdmB the in DnrK (Fig. of the of RdmB with The structure of the complex is similar to that of the complex as by a of for the in the In particular, the of and of the is RdmB is to a of small molecule methyltransferases as by striking in amino acid sequence and three-dimensional structure (11Jansson A. Niemi J. Lindqvist Y. Mäntsälä P. Schneider G. J. Mol. Biol. 2003; 334: 269-280Crossref PubMed Scopus (33) Google Scholar), the enzyme does not a methyltransferase reaction. Here, we have shown that RdmB catalyzes a hydroxylase reaction where a hydroxyl group derived from molecular oxygen is into the of the anthracycline substrate. are that have to be is RdmB not a methyltransferase, and is a mechanism for this hydroxylation The in amino acid sequence the site RdmB and its closest homologue, DnrK, which catalyzes methyl is the of (DnrK) by the However, is not in the methyltransferase reaction catalyzed by DnrK as the of this amino acid with by mutagenesis results in an active methyltransferase (12Jansson A. Koskiniemi H. Mäntsälä P. Niemi J. Schneider G. J. Biol. Chem. 2004; 279: 41149-41156Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The in RdmB on the also leads to a mutant to the hydroxylation of any methyltransferase the structural basis for the of RdmB to methyl transfer has to be In a methyl transfer reaction using a the methyl group has to be in with the oxygen of the substrate as as the atom of AdoMet for the reaction to This is the in DnrK (12Jansson A. Koskiniemi H. Mäntsälä P. Niemi J. Schneider G. J. Biol. Chem. 2004; 279: 41149-41156Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). However, in the active site of RdmB, the AdoMet is relative to the substrate with the that the methyl group of AdoMet in a not for methyl transfer (Fig. The in the of the cofactor AdoMet is because of in which are also with structural can be to large changes the enzymes in sequence and structure of the and amino in This is the only particular structural the ternary of RdmB and In RdmB, it has the substrate binding with It be to that be by decarboxylation of or similar anthracycline followed by as was in (7Niemi J. Mäntsälä P. J. Bacteriol. 1995; 177: 2942-2945Crossref PubMed Google Scholar, 14Wang Y. Niemi J. Airas K. Ylihonko K. Hakala J. Mäntsälä P. Biochim. Biophys. Acta. 2000; 1480: 191-200Crossref PubMed Scopus (19) Google Scholar). of an carbon in antibiotic biosynthesis usually by the of a enzyme Dickens M.L. Priestley N.D. Strohl W.R. J. Bacteriol. PubMed Google Scholar), in this the reaction was shown to in vitro in the presence of RdmB, a methyltransferase The data in this can be to the mechanistic shown in The evidence that the substrate of the reaction is DmaA, a acid that at pH is present in the as for by in the in the pH system used in the of is the first because the observed intermediate the The decarboxylation step results in the formation of a and However, this is not a substrate of RdmB and is not an intermediate of the reaction. the enzyme of the carbanion by of the charge through resonance the aromatic ring system of the The delocalization of the electrons into the aromatic ring system is further facilitated by the positive charge of the AdoMet cofactor. This is by the that an AdoMet analogue a positive charge at the group at pH (Fig. G. J. Mol. Biol. 1997; PubMed Scopus Google Scholar), also whereas the AdoMet analogue does not act as a cofactor. The structure of RdmB in complex with sinefungin that the ligand is in the manner as AdoMet, that the positive charge by is at a similar in the active site. The delocalization of electrons into the can be used for the activation of oxygen (Fig. are a the C-10 of the product that could the oxygen molecule (Fig. the reaction The intermediate observed in the absence of thiol has a mass consistent with a particularly in the reaction with as its UV-visible is the is to be in the aromatic of the This to the intermediate a which is to the hydroxyl group by the thiol A not to be in this because the mutant The to the of In the are in the the insertion of oxygen is a and hydroxylases usually a metal or or an to activate oxygen. In the cofactor an from the to the oxygen to a After the is into a intermediate J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). This the reaction catalyzed by RdmB where the ring system to be part of the substrate A in the of the atom of the substrate can facilitate the binding of oxygen for the formation of a as in the reaction with The is to a hydroxyl group and by the thiol and not a on the A mechanism for this step to the 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) can be mechanism for the hydroxylation reaction catalyzed by RdmB could the formation of a AdoMet as in the of enzymes 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, Chem. Rev. 2003; Scopus Google Scholar). of the which AdoMet as a of a in the of the cofactor binding site 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). However, RdmB is not a of this enzyme It lacks the and does not any Therefore, a mechanism as suggested for the enzyme for In the ability of the AdoMet analogue sinefungin to AdoMet in the reaction also a mechanism. have shown that the hydroxylation reaction catalyzed by the methyltransferase homologue RdmB does not cofactors usually associated with activation of molecular oxygen such as flavins or metal the enzyme AdoMet as a cofactor for the activation of a novel function of this cofactor.