Purification , Characterization , and Kinetic Mechanism of S-Adenosyl-L-methionine : Macrocin 0-Methyltransferase from Streptomyces fradiae ”

5‘-Adenosyl-L-methionine:macrocin O-methyltransferase catalyzes conversion of macrocin to tylosin, the terminal and main rate-limiting step of tylosin biosynthesis in Streptomyces fradiae. The O-methyltransferase was stabilized in vitro and purified to electrophoretic homogeneity. The purified enzyme had a molecular weight of 65,000 and consisted of two identical subunits of 32,000 with an isoelectric point of 4.5. The enzyme required MgZ+, Mn2+, or Coz+ for maximal activity and was catalytically optimal at pH 7.5-8.0 and 31 “C. The 0-methyltransferase catalyzed the conversion of macrocin to tylosin at a stoichiometric ratio of 1:l. The enzyme also mediated conversion of lactenocin -., desmycosin. The corresponding Vm,JKm ratios for the two analogous conversions were similar, and both enzymic conversions were susceptible to extensive competitive and noncompetitive inhibitions by macrolide metabolites. Steady-state kinetic studies for initial velocity, substrate analogue, and product inhibitions have allowed formulation of Ordered Bi Bi as the reaction mechanism for macrocin 0-methyltransferase.

Tylosin, a 16-membered macrolide antibiotic produced by Streptomyces fradiae, is used in the swine industry as a growth promotant and in veterinary medicine for treatment of infections caused by Gram-positive bacteria and mycoplasma. The biosynthesis of tylosin and its general regulation have been of considerable scientific as well as industrial interest for over a decade (1,2). Recent information on the biosynthetic pathway of tylosin (1, 3) makes it feasible to study regulation of tylosin biosynthesis at the enzyme level. Knowledge gained in such study may be applicable to tylosin yield improvement.
We chose to examine S-adenosyl-L-methionine:macrocin 0methyltransferase initially since this enzyme catalyzes the terminal and major rate-limiting step ( Fig. 1) of tylosin biosynthesis in s. fradiue (4.5). Preliminary studies of macrocin 0-methyltransferase from cell-free extracts of S. fradiue indicated that the specific activity of the enzyme was higher in mutants producing higher levels of tylosin ( 5 ) and that the enzyme was inhibited by tylosin and some of its precursors and shunt metabolites (6). However, the molecular nature of the rate-limiting reaction is still unknown. Plausible molecular mechanisms that are amenable to an enzymological investigation might be inadequate enzyme levels (5), metabolic inhibition (6), deficiency of cosubstrate (i.e. methyl group * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. donor), or enzyme degradation. In this report,' we describe an effective purification of macrocin 0-methyltransferase to electrophoretic homogeneity. We show that the purified enzyme has a narrow substrate specificity and is subject to broad metabolic inhibitions by two distinctive kinetic patterns. In addition, our kinetic data strongly suggest that macrocin 0methyltransferase follows a compulsory ordered reaction mechanism.

EXPERIMENTAL PROCEDURES AND RESULTS'
Substrate Specificity-In addition to macrocin, the O-methyltransferase was capable of catalyzing 3"'-O-methylation of 20-dihydromacrocin, lactenocin, and 20-dihydrolactenocin (Table 111) with AdoMet3 as the methyl-group donor. The enzyme activity with either form of macrocin was similar to that with either form of lactenocin. No enzymic methylation with demethylmacrocin, demethyllactenocin, the four macrolide intermediates preceding demethylmacrocin in the preferred pathway (3), and tylactone was HPLC-detectable when each macrolide compound was examined for its disappearance and product formation under the optimal catalytic conditions of the enzyme. Among the six methyl-containing compounds tested (Table 111), only AdoMet could serve as the methylgroup donor in macrocin 0-methyltransferase-catalyzed methylation of macrocin or lactenocin.
Enzyme Inhibition-Macrocin O-methyltransferase-catalyzed conversion of macrocin to tylosin was inhibited moderately by demethyllactenocin or demethylmacrocin and weakly by desmycosin or relomycin (Table IV). Similarly, macrocin 0-methyltransferase-catalyzed conversion of Iactenocin to desmycosin was inhibited moderately by demethyllactenocin or demethylmacrocin and weakly by tylosin or relomycin. No substrate inhibition of the 0-methyltransferase by macrocin or lactenocin was shown at a concentration up to 120 wM.
Both macrocin 0-methyitransferase-catalyzed conversions were inhibited weakly by AdoHcy and strongly by sinefungin ' An initial work of this paper was presented (by W. K   Tested at 250 pM with 40 pM macrocin. or A9145C (Table V). AdoMet, the cosubstrate of either conversion, was not inhibitory at a concentration up to 1 mM.

DISCUSSION
Purification-Addition of phenylmethylsulfonyl fluoride, AdoMet, and ethanol to cell-free extracts of S. fradiue can protect macrocin 0-methyltransferase from inactivation for a minimum of 1 week at 4 "C. Under the stabilizing conditions, the 0-methyltransferase has been purified to apparent electrophoretic homogeneity by a simple four step chromatographic procedure ( Table I). Protection of the O-methyltransferase from inactivation by phenylmethylsulfonyl fluoride and improvement of its stability by each of the four chromatographic steps indicate that enzyme lability is likely caused by a serine motease(s). Purification of macrocin O-methyltrans-Success in isolation of biosynthetic enzymes from other macrolide pathways has been limited (2,16).
Physical and Catalytic Properties-Macrocin O-methyltransferase has a molecular weight of 65,000 and a subunit size of 32,000; therefore, the native enzyme appears to be dimeric. Automated sequence analysis by Edman degradation of the purified enzyme revealed a single 34-residue aminoterminal sequence (17), suggesting that the O-methyltransferase consists of two identical subunits. The subunit identity is substantiated by the observation that an oligonucleotide probe constructed according to a part of the terminal sequence is specific for a single DNA sequence, which when cloned, restored the 0-methyltransferase activity to a macrocin 0methyltransferase-negative mutant of S. fradiae (17,18). Macrocin 0-methyltransferase has no activity in the absence of a metal ion, and maximal activity can be shown with Mg+, Mn2+ or Co2+. Possibly, an intrinsic divalent metal ion binds loosely to the 0-methyltransferase in uiuo and dissociates readily from the enzyme during the enzyme purification.
Substrate Specificity-The activity data (Table III) and kinetic constants (as described under "Initial Velocity Pattern" of the Miniprint) for the 0-methyltransferase indicate that the enzyme catalyzes conversion of macrocin to tylosin and of lactenocin to desmycosin at an approximately equivalent efficiency. Such substrate specificity toward macrocin and lactenocin is in agreement with 3"'-O-methylation of an attached 2"'-O-methylated 6-deoxy-D-allose as indicated from previous biochemical studies of S. fradiae (3-5). Whether macrocin 0-methyltransferase or an independent O-methyltransferase can catalyze 3-0-methylation of free 2-0-methylated 6-deoxy-~-allose, presumably as a dTDP-derivative, remains to be determined. This enzymic methylation is implicated in formation of mycinose from another proposed route of tylosin biosynthesis (i.e. mycaminosyltylonolide +mycinose demycarosyltylosin +mycarose tylosin) in S.
ferase, aiylosin biosynthetic enzyme from the preferred pathfradiae (1,19).   nd, not detectable (i.e. k2% of either uninhibitory activity). e -, not analyzed. chromatography of a S. fradiae extract. The methylation activity peak with macrocin as the macrolide substrate was inseparable from that with lactenocin as the macrolide substrate (data not shown), thus a single 0-methyltransferase appears responsible for both macrocin + tylosin and lactenocin + desmycosin conversions. Neither demethylmacrocin nor demethyllactenocin is a macrolide substrate for macrocin 0-methyltransferase and, as described in the following paper (20), the two compounds are macrolide substrates for a sepa-rate demethylmacrocin 0-methyltransferase. Therefore, the 2"'-O-methylation of bound B-deoxy-~-allose as catalyzed by demethylmacrocin 0-methyltransferase is the prerequisite of the subsequent 3'"-O-methylation as catalyzed by macrocin 0-methyltransferase, and this methylation order is consistent with the isolation of two distinguishable O-methyltransferasedeficient mutants from 5'. fradiae as described previously (5,6).
Macrolide and Nucleoside Inhibitions-The range and extent of macrolide inhibitions for macrocin O-methyltransferase-catalyzed conversion of macrocin to tylosin are indistinguishable from those for macrocin O-methyltransferase-catalyzed conversion of lactenocin to desmycosin (Table IV). The inhibition of macrocin 0-methyltransferase by tylosin, desmycosin, or relomycin (i.e. C-20 reduced form of tylosin) is characteristic of product inhibition (6), which has also been demonstrated for demethylmacrocin 0-methyltransferase (20) and erythromycin C 0-methyltransferase (21). The observation that both demethylmacrocin and demethyllactenocin are the most potent macrolide inhibitors of macrocin 0methyltransferase (Tables IV and VI) is unique for an 0methyltransferase, since either metabolite of tylosin biosynthesis also serves as a macrolide substrate for demethylmacrocin 0-methyltransferase (20). A similar broad inhibition of macrocin 0-methyltransferase from cell-free extracts of S. frudiae by macrolide compounds has been reported (4). We speculate that macrocin 0-methyltransferase is subject to in vivo regulation by broad but selective metabolic inhibition rather than specific product inhibition. The structural requirement of a macrolide compound for inhibition of macrocin 0-methyltransferase has not been defined; however, all five inhibitory macrolide compounds contain a mycaminosyltylactone core that is connected to 6-deoxy-D-allose or mycinose (Table IV) as was pointed out previously (6). Presence of mycarosyl moiety or reduction at the C-20 position of tylactone has little effect on the enzyme inhibition.
The inhibition by AdoHcy and no inhibition by its D-isomer (Table V) indicates a high specificity of the nucleoside reaction product toward macrocin 0-methyltransferase. Sinefungin and A9145C are much more inhibitory than AdoHcy in macrocin 0-methyltransferase-catalyzed methylation of   Kinetic Patterns and Reaction Mechanism-Since the 0methyltransferase catalyzes stoichiometric conversion of macrocin to tylosin (Fig. 4) or of lactenocin to desmycosin (data not shown), no shunt metabolic route was operative for either conversion under the reaction conditions. Separability of tylosin and desmycosin from each other and from other macrolide compounds (as substrate, inhibitor, and/or internal standard) by the HPLC procedure (9) permits highly reproducible (i.e. &2% between duplicate analyses) determination of the enzyme activity. Macrocin O-methyltransferase-catalyzed formation of tylosin (or desmycosin) from macrocin (or lactenocin) is linear with time within 10 min from reaction initiation and thus provides a reliable representation of initial activity (i.e. initial velocity) in our kinetic studies.
The kinetic patterns that were used in determining a kinetic mechanism for macrocin 0-methyltransferase-catalyzed methylation are summarized in Table VI. In the absence of an inhibitor, the intercepting substrate-interaction pattern for macrocin 0-methyltransferase-catalyzed conversion of macrocin to tylosin indicates that the enzyme follows a sequential mechanism (26); i.e. both macrocin and AdoMet must bind to the 0-methyltransferase prior to release of either tylosin or AdoHcy from the enzyme. The binding order of the two substrates can be derived from the inhibition patterns of demethylmacrocin and sinefungin (Table VI), which are presumed to be dead-end inhibitors of macrocin O-methyltransferase. The inhibition of the 0-methyltransferase by demethylmacrocin and sinefungin is competitive with regard to macrocin and AdoMet, respectively. Thus, the uncompetitive inhibition by demethylmacrocin with respect to AdoMet and the noncompetitive inhibition by sinefungin with respect to macrocin become diagnostic of a binding order of the 0methyltransferase with AdoMet as the leading substrate (13).
Consistent with this binding order, the competitive inhibition with variable macrocin and the uncompetitive inhibition with variable AdoMet are also revealed by demethyllactenocin. The order of release of tylosin and AdoHcy from macrocin 0methyltransferase can be derived from the inhibition patterns of desmycosin and AdoHcy (Table VI). The inhibition of the 0-methyltransferase by AdoHcy is competitive with regard to AdoMet and noncompetitive with regard to macrocin. Desmycosin was used as a macrolide product analogue in the inhibition study of macrocin 0-methyltransferase-catalyzed conversion of macrocin +tylosin, so that the enzyme activity could be determined reproducibly by monitoring tylosin formation with HPLC. The inhibition of the O-methyltransferase by desmycosin is noncompetitive with respect to either macrocin or AdoMet. Since either desmycosin or tylosin is a weak macrolide inhibitor (Tables IV and VI) and either macrocin or lactenocin is an effective macrolide substrate (Table 111), the same noncompetitive inhibition pattern of the 0-methyltransferase by desmycosin may apply to both macrocin + tylosin and lactenocin + desmycosin conversions. That this analysis appears to be the case is consistent with the noncompetitive inhibition of the macrocin O-methyltransferase-catalyzed conversion of lactenocin + desmycosin by tylosin (also used as a macrolide product analogue) with regard to lactenocin. The product inhibition patterns not only substantiate the binding order of macrocin 0-methyltransferase with AdoMet as the leading substrate but also support an order of release from the enzyme with AdoHcy as the last product. From all kinetic patterns as described above, we conclude that macrocin 0-methyltransferase-catalyzed methylation of macrocin follows Ordered Bi Bi (Scheme 1) as the kinetic reaction mechanism. The intercepting substrate interaction pattern with lactenocin as the macrolide substrate (data not shown) and the noncompetitive inhibition of the lactenocin + desmycosin conversion by tylosin suggest that macrocin 0-methyltransferase-catalyzed methylation of lactenocin follows an analogous Ordered Bi Bi reaction mechanism. Metabolic Inhibition of Macrocin 0-Methyltransferme as Regulation of Tylosin Biosynthesis-Macrocin O-methyltransferase is subject to metabolic inhibition by both competitive and noncompetitive patterns with respect to macrocin (Table  VI). Thus, demethylmacrocin or demethyllactenocin (macrolide intermediate) may exhibit a moderate inhibitory effect at the active site of the enzyme. In contrast, tylosin or desmycosin (macrolide reaction product) or presumably relomycin (tylosin analogue and shunt metabolite) may exert a weak inhibitory effect at a regulatory site of the enzyme. The broad metabolic inhibition of macrocin 0-methyltransferase could be a factor to the rate-limiting conversion of macrocin to tylosin and may play a regulatory role in tylosin biosynthesis of S. fradiae. Regardless of either inhibition mode, significance of the metabolic regulation might be overcome by increasing the 0-methyltransferase activity via a "gene cloning" approach, which is being pursued (17, 18). The specific inhibition by tylosin, the major macrolide product, possibly at a regulatory site of macrocin 0-methyltransferase provides a practical model for yield improvement of this industrially important antibiotic by site-directed modification of the enzyme. a competlllve. noncompel~l~ve or uncompetltlve Inh#blllon pattern The data were then llned lo  migrated as a single profem band (Fig 2).