©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Substrate Channeling in the Lumazine Synthase/Riboflavin Synthase Complex of Bacillus subtilis(*)

Klaus Kis , Adelbert Bacher

From the (1)Department of Organic Chemistry and Biochemistry, Technical University of Munich, Lichtenbergstra4, D-85747 Garching, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 subunits surrounding a core of three subunits. The subunits catalyze the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (PYR) with 3,4-dihydroxy-2-butanone 4-phosphate (DHB) yielding 6,7-dimethyl-8-ribityllumazine. This intermediate is converted to riboflavin by the subunits via an unusual dismutation. The second product of this reaction is PYR, which is also a substrate of the subunits and can be recycled in the catalytic process.

Sigmoidal kinetics would be expected for the formation of riboflavin from PYR and DHB and are indeed observed with mixtures of artifactual capsids and subunit trimers. In contrast, the formation of riboflavin from PYR and DHB by the native is characterized by a finite initial rate, which is similar to the rate of lumazine formation. Most notably, the rate of riboflavin formation has its maximum value at t = 0 and decreases dramatically after the consumption of PYR and DHB despite the presence of transiently formed lumazine. These data suggest that a significant fraction of DHB is converted to riboflavin by substrate channeling, which is conducive to an improved overall catalytic rate of riboflavin formation at low substrate concentrations. The channel is leaky, and the intermediate lumazine is therefore transiently accumulated in the bulk solution. The partitioning factor relating the direct formation of riboflavin via substrate channeling and the formation of transient 6,7-dimethyl-8-ribityllumazine increases at low concentrations of the substrates PYR and DHB and has a maximum value at pH 7.5. Channeling appears to result from the compartmentalization of the subunits inside the icosahedral subunit capsid whose catalytic sites are located close to the inner capsid surface.


INTRODUCTION

The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of 3 subunits and 60 subunits (Bacher et al., 1980). The 1-MDa protein complex catalyzes the terminal two reaction steps in the biosynthesis of riboflavin (Neuberger and Bacher, 1986; Volk and Bacher, 1990; Kis et al., 1995). More specifically, the subunits catalyze the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (PYR)()(Fig. 1) with 3,4-dihydroxy-2-butanone 4-phosphate (DHB) under formation of 6,7-dimethyl-8-ribityllumazine (LUM) and orthophosphate. The subunits catalyze the dismutation of the lumazine substrate yielding riboflavin and the pyrimidine PYR, which serves both as a product of the subunits and also as a substrate of the subunits. Thus the pyrimidine produced by the catalytic action of the subunits can be recycled by the subunits. The overall stoichiometry implicates the formation of one molecule of riboflavin and two molecules of inorganic phosphate from one molecule of PYR and two molecules of DHB. Every second molecule of PYR must be processed twice by the subunits of the enzyme complex in order to allow for quantitative conversion of PYR to the product, riboflavin.


Figure 1: Biosynthesis of riboflavin. , 6,7-dimethyl-8-ribityllumazine synthase; , riboflavin synthase.



The reaction mechanism of the lumazine synthase ( subunit) has been studied in some detail (Kis et al., 1995). It appears likely that the initial reaction step consists in the formation of a Schiff's base by reaction of the 5-amino group of PYR with the carbonyl group of DHB. Formation of the imine motif may facilitate the abstraction of a proton from position 3 of DHB, and the resulting carbanion could eliminate phosphate. The resulting intermediate could cyclize under formation of LUM.

The riboflavin synthase reaction ( subunit) has been studied in considerable detail by Plaut and co-workers using the enzyme from bakers' yeast. This elegant work has been reviewed repeatedly (Plaut et al., 1970; Plaut and Harvey, 1971; Plaut and Beach, 1975; Bacher, 1990).

The structure of the complex of B. subtilis has been studied in considerable detail (Bacher et al., 1986; Ladenstein et al., 1986, 1994; Bacher and Ladenstein, 1990; Ritsert et al., 1995). The 60 subunits form an icosahedral capsid with triangulation number T = 1, in which all protomers occupy equivalent positions. The central core of the icosahedral capsid is occupied by a trimer of subunits. Dissociation of the enzyme complex under mild conditions (e.g. Tris hydrochloride, pH 8) is conducive to the formation of subunit trimers and of large, heterogeneous aggregates consisting of more than 100 subunits, which may represent incomplete icosahedral structures with triangulation number T = 3 or 4 (Bacher et al., 1986). In the presence of substrate analogs such as 5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, these aggregates can be converted to hollow, icosahedral capsids of 60 subunits, which differ from the native enzyme by the absence of subunits from the central core space (Schott et al., 1990a).

More recently, it was found that the overexpression of subunits in a recombinant Escherichia coli strain is conducive to the formation in vivo of hollow capsids.() The artifactual capsids catalyze the formation of LUM from PYR and DHB at virtually the same rate as the native enzyme complex (Kis et al., 1995). Moreover, it was found that even the large, heterogeneous subunit aggregates with molecular masses in the range of 3-5 MDa catalyze the formation of LUM. However, it is possible that the catalytic activity of these aggregates depends on ligand-induced reaggregation conducive to the formation of capsids.

X-ray structures for the icosahedral subunit capsid have been obtained with crystals of the native capsids and with artifactual capsids obtained by ligand-driven renaturation of subunits (Ladenstein et al., 1988, 1994; Ritsert et al., 1995). The two respective structures are virtually identical, but the artifactual capsids diffract x-rays to significantly higher resolution than the native species.

The subunit capsid is best described as an assembly of 12 densely packed pentamers. Channels formed by five skew helices surround the 5-fold axes. The 60 equivalent active sites are located at the interface between adjacent pentamer subunits in close proximity of the inner capsid surface. Since the capsid is rather densely packed, it is as yet an open question how the substrates and products can exchange with the bulk solvent. The channels along the five-fold axes could allow the passage of substrates but appear too narrow for the passage of LUM and of riboflavin (Ladenstein et al., 1994).

Unfortunately, the crystals of the complex yield no structural information for the subunits. It appears likely that the subunits can be located on any of the 10 three-fold axes of the icosahedral capsid. This would allow for 20 different positions with respect to the crystal lattice. Sequence analysis has suggested that each subunit forms two homologous domains (Schott et al., 1990a). Moreover, NMR studies suggest that the subunit trimer may not obey strict C symmetry.() However, the trimers might be characterized by pseudo-C or pseudo-D symmetry.

Preliminary studies by Neuberger and Bacher(1986) had indicated that the complex has unexpected kinetic properties. This paper describes a more detailed analysis of steady state kinetics indicative of substrate channeling between the and subunit sites.


EXPERIMENTAL PROCEDURES

Materials

5-Nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (Cresswell et al., 1960) and 6,7-dimethyl-8-ribityllumazine (Bacher, 1986) were prepared by published procedures. PYR was freshly prepared by hydrogenation of 5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione over palladium on charcoal in aqueous solution (Sedlmaier et al., 1987). Both enantiomers of DHB were prepared as described elsewhere (Kis et al., 1995).

Proteins

The lumazine synthase/riboflavin synthase complex (``heavy riboflavin synthase'') was purified from the derepressed B. subtilis mutant H94 (Schott et al., 1990b). Recombinant capsids were kindly provided by Markus Fischer and Karl Kugelbrey.

Estimation of Protein Concentration

Protein concentration was monitored photometrically using absorbance coefficients of = 7.0 for the lumazine synthase/riboflavin synthase complex (Bacher et al., 1980) and of = 8.0 for isolated subunits (Bacher et al., 1986).

Enzyme Assay

Reaction mixtures contained DHB (S or R) and freshly prepared PYR in the concentrations indicated. The mixtures also contained 100 mM potassium phosphate, pH 7.0, 2 mM EDTA, and 2 mM dithiothreitol unless otherwise noted. They were incubated at 37 °C. Aliquots were taken at intervals, and the reaction was quenched by the addition of trichloroacetic acid to a final concentration of 0.4 M. Concentrations of LUM and riboflavin were determined by high performance liquid chromatography analysis. Columns of Nucleosil RP18 (4 250 mm) were used throughout. The effluent was monitored fluorometrically. For the analysis of LUM, an eluent containing 7% methanol and 30 mM formic acid was used (excitation, 408 nm; emission, 487 nm). The retention volume was 8.4 ml. For the analysis of riboflavin, the eluent contained 40% methanol and 100 mM ammonium formate (excitation, 445 nm; emission, 516 nm). The retention volume was 6.0 ml.

In experiments with the complex, the total amount of lumazine formed by the catalytic activity of the subunits was calculated as the sum of the molar amount of LUM plus 2 times the molar amount of riboflavin to account for the stoichiometry of the dismutation reaction, which generates one molecule of riboflavin from two molecules of LUM.

Calculations

Numerical simulations were performed using the program Microsoft Excel Solver on a PC with a 386DX processor from Intel and a 387DX coprocessor from Cyrix.


RESULTS

The lumazine synthase/riboflavin synthase complex catalyzes two consecutive reactions as shown in Fig. 1. Steady state kinetic studies of the second reaction step (i.e. the formation of riboflavin from the intermediate LUM) have been reported earlier (Bacher et al., 1980). More recently, kinetic studies of the first reaction step, i.e. the formation of the lumazine LUM from PYR and DHB have been performed (Kis et al., 1995). Steady state kinetic parameters of both partial reactions are summarized in . Kinetic parameters for subunit trimers (``light riboflavin synthase'') and for recombinant capsids devoid of subunits are also shown. It should be noted that the K value for LUM of the complex is about 10 times larger as compared to light riboflavin synthase ().

The formation of riboflavin from the substrates PYR (initial concentration, 800 µM) and DHB (initial concentration, 105 µM) by the complex is shown in Fig. 2. The initial reaction phase is characterized by the formation of riboflavin at a rate of 2,600 nmol mg h, whereas the formation of the intermediate, LUM, proceeds with an apparent rate of 6,100 nmol mg h. After the consumption of the substrate DHB, the concentration of the transient intermediate LUM shows a slow decrease. Concomitantly, the velocity of riboflavin formation decreases by a factor of about 30 as compared to the initial reaction phase. This kinetic behavior is unexpected. Most notably, the initial rate of the second reaction step in a sequential reaction would be expected to have a value of zero, and the time course of riboflavin formation would be expected to have a sigmoidal character. On the contrary, the rate of riboflavin formation by the complex has its maximum value at t = 0 and shows first a slow decrease and later, when the substrate DHB has been consumed, a very marked decrease. Numerically, the initial rate of riboflavin formation in this experiment corresponds to 1.3 times the value of V for the conversion of exogenous LUM into riboflavin by the complex.


Figure 2: Kinetics of product formation by the complex. A, formation of LUM () and riboflavin () from S-DHB and PYR. B, velocity of riboflavin formation. The reaction mixture contained 800 µM PYR, 105 µMS-DHB, and 74 µg of the complex in 1 ml of potassium phosphate buffer (100 mM, pH 7.0) at 37 °C. The reaction was started by the addition of protein. The lines were calculated as described under ``Results'' using the refined parameters in Table III.



The reaction catalyzed by the subunit is a bisubstrate reaction. Thus, this reaction step is inherently first order for each of the substrates PYR and DHB. However, the experiment described above can be treated as zero order for the pyrimidine for the following reasons. (i) The pyrimidine was present in an 8-fold excess over the carbohydrate. (ii) The K for the pyrimidine (5 µM) is small as compared to the actual pyrimidine concentration (800 µM at t = 0). (iii) The second reaction step, i.e. the formation of riboflavin by the subunit, regenerates a certain amount of PYR as the second product of the dismutation reaction, thus slowing down the consumption of PYR.

As an approximation, the formation of lumazine can be therefore treated as a one substrate reaction depending on the concentration of DHB. The velocity of lumazine formation is given by Equation 1,

On-line formulae not verified for accuracy

where [DHB] is the concentration of the substrate DHB at time t, K is the Michaelis constant of the complex for the substrate DHB (130 µM), and V is the maximum velocity of formation of LUM.

The reaction catalyzed by the subunits, i.e. the dismutation of the lumazine under formation of riboflavin and the pyrimidine PYR, is also a bisubstrate reaction. Surprisingly, this reaction has been shown to follow Michaelis-Menten kinetics over a wide range of concentrations in experiments by Harvey and Plaut(1966) using the enzyme from bakers' yeast. Later, the subunit trimer (light riboflavin synthase) of B. subtilis has also been shown to obey Michaelis-Menten kinetics over a wide range of substrate concentrations (Bacher et al., 1980). Thus, the velocity of riboflavin formation from LUM can be given as

On-line formulae not verified for accuracy

where [LUM] is the concentration of LUM, V is the maximum velocity of riboflavin formation, and K is the Michaelis constant of the complex for LUM (130 µM).

Using the kinetic parameters for the two reaction steps of the complex given in and the starting conditions of the experiment in Fig. 2, the formation of riboflavin by the sequential reaction DHB + PYR LUM RIB can be predicted by numerical integration of Equations 1 and 2. A comparison of the numerical prediction with the experimental data is shown in Fig. 3. It is obvious from this comparison that the model of independent, consecutive reactions as specified by Equations 1 and 2 is not an appropriate description of the experimental data. Most notably, the absence of a lag phase for the experimentally observed formation of riboflavin indicates that the formation of riboflavin does not depend on the presence of the intermediate LUM in finite concentration in the bulk solvent. This could be explained by the hypothesis that a significant fraction of the subunit product is directed to the subunit active site by substrate channeling rather than by equilibration with the bulk solution (for a more detailed definition of substrate channeling, see ``Discussion'').


Figure 3: Formation of riboflavin by the complex by substrate channeling (solid line, data from Fig. 2) and prediction of riboflavin formation by the free diffusion model (dashed line, calculated with Equations 1 and 2 and kinetic constants from Table I).



The transient formation of LUM in substantial concentration also indicates that only a fraction of the intermediate LUM is handled by substrate channeling, whereas the remaining part reacts via the standard consecutive model after equilibration with the bulk solvent. In other words, we propose partitioning between a channeled pathway and a pathway involving equilibration with the solvent, (i.e. a ``leaky channel'' model following the terminology of Ovádi (1991)).

It appeared likely at this stage of the analysis that the kinetic anomalies are a direct consequence of the tight physical association of and subunits in the complex. This hypothesis can be tested directly, because the subunits as well as the subunits can assemble separately to yield catalytically active oligomers. Thus, subunits can form stable trimers (light riboflavin synthase), which have approximately 1.7 times the turnover number as the complex and a K value of 13 µM as compared to 130 µM for the complex. Moreover, the recombinant expression of subunits in E. coli in the absence of subunits yields icosahedral particles consisting of 60 subunits whose V and K values are similar to the complex () (Kis et al., 1995).

Fig. 4shows a reconstruction experiment using molecules and recombinant molecules at a molar ratio of 1:1 (i.e. 3 subunits/60 subunits as in the native complex). The concentrations of LUM and of riboflavin are shown. Most notably, the experiment shows the expected lag phase with a value of zero for the initial rate of riboflavin formation.


Figure 4: Kinetics of product formation by a 1:1 mixture of and molecules. A, formation of LUM () and riboflavin () from PYR and DHB. B, velocity of riboflavin formation. The reaction mixture contained 600 µM PYR and 230 µMS-DHB in 100 µl of 100 mM potassium phosphate, pH 7.0. The reaction was started by the addition of 34.2 µg of recombinant lumazine synthase () and of 2.5 µg of light riboflavin synthase (). The lines were calculated as described under ``Results'' using the refined parameters from Table II.



Using Equations 1 and 2, the formation of riboflavin by the / mixture (Fig. 4) was simulated using the values of V and K for and molecules as shown in . Subsequently, the values for V and K were refined by numerical iteration. A close fit was obtained with the refined parameters given in . The lines in Fig. 4A show the numerical fit. Also shown is the calculated velocity of riboflavin formation (Fig. 4B). The fundamental difference between the experiments in Fig. 2and Fig. 4is best explained by the involvement of substrate channeling in the enzyme complex but not in the mixture.

A simplified kinetic network for the partial reactions catalyzed by the complex is shown in Fig. 5. We assume that the transfer of the lumazine LUM from the subunits to the subunits can either occur by equilibration of the intermediate with the bulk solvent by free diffusion, or by substrate channeling without equilibration with the bulk solution. The simplified scheme does not take into account that the two lumazine molecules required for the dismutation reaction must be supplied to the subunits one by one. Moreover, the dissociation of products from the subunits as well as the binding of substrate to the subunits may be more complex than expressed in the scheme.


Figure 5: Kinetic model of the reactions catalyzed by the complex. , subunits; , subunits. Enzyme-bound ligands are shown in parentheses; RIB, riboflavin.



Even for the simplified scheme, the rate constants for most of the partial reactions shown are unknown. It is therefore not possible at this time to perform a comprehensive numerical simulation of the proposed leaky channel behavior of the complex. However, a successful attempt was made to simulate the data in Fig. 2by a heuristic kinetic model. It is obvious that the contributions of the channeled and the free diffusion pathway must be additive. We assume that the free diffusion pathway can be described, in principle, by Equations 1 and 2. Moreover, we assume that, under a given set of starting conditions, each lumazine molecule formed after the initiation of the reaction has a finite probability to enter the channeled or the unchanneled pathway. This probability of a molecule to react via the channel is given by the partitioning factor defined as shown by Equation 3,

On-line formulae not verified for accuracy

where [RIB] is the concentration of riboflavin and [LUM] is the apparent concentration of LUM, i.e. the concentration of the transient intermediate in Fig. 2. The factor 2 must be introduced because two lumazine molecules must enter the channeled pathway in order to obtain one molecule of riboflavin. The experimental value of at t = 0 is easily determined from the initial velocities in Fig. 2. The formation of riboflavin via channeling is rigidly coupled to the velocity of the first reaction step and can be given by Equation 4.

On-line formulae not verified for accuracy

The formation of transient lumazine can be given by Equation 5.

On-line formulae not verified for accuracy

The formation of riboflavin via free diffusion can be described by Equation 6.

On-line formulae not verified for accuracy

The total riboflavin formation rate results from the contributions of the reactions via the channel and via free diffusion, respectively. Thus, we obtain Equation 7.

On-line formulae not verified for accuracy

The total formation of riboflavin is described by the differential Equations 4 and 6, which can be integrated numerically. The integral was fit to the experimental data in Fig. 2by numerical iteration yielding the refined parameters shown in I. The lines in Fig. 2A represent the calculated values using these refined parameters. The agreement between the numerical simulation and the experimental data is excellent. The rate of riboflavin formation as calculated by this numerical approach is shown in Fig. 2B.

In the experiment with the complex, rate acceleration by substrate channeling can of course only occur while the substrates PYR and DHB are available. After their exhaustion, the consumption of the transient lumazine LUM occurs by the unchanneled reaction. Since the K of the complex for LUM is relatively large as compared to free trimers, the consumption of the transient LUM is slow in the experiment with the complex (Fig. 2) as compared to the / mixture (Fig. 4).

The partitioning factor depends on the substrate concentration. For a systematic study, the initial concentration of PYR or of DHB was varied, while the other substrate was proffered at a high concentration well above the respective K value (Fig. 6). In both experiments, the partitioning factor increased at lower substrate concentrations. Values close to 0.9 were observed at low concentration of the pyrimidine PYR, even in the presence of DHB at high concentration. Values of close to 1 can be expected when both substrates are present at low concentrations.


Figure 6: Partitioning coefficient derived from initial rates according to Equation 3 at different substrate concentration. The reaction mixtures contained 1.5 mM DHB (A) or 2 mM PYR (B) and 1.5 µg of complex in 100 µl of 100 mM potassium phosphate buffer, pH 7.0.



The data indicate that substrate channeling is favored by a low velocity of lumazine formation. This suggested that a substrate analog for lumazine synthase with an inherently low conversion rate should be conducive to efficient substrate channeling. We have found earlier that the R-enantiomer of DHB is converted to LUM with a velocity of about 1/6 as compared to the natural S-enantiomer. Fig. 7shows an experiment where the R- and S-enantiomers were converted to riboflavin by the complex under identical conditions. Numerical modeling was performed as described above. The partitioning factor is 0.4 in the case of the S-enantiomer and 0.6 in the case of the R-enantiomer. The initial rate of riboflavin formation is of course lower with the R-enantiomer due to its slow conversion into the intermediate LUM.


Figure 7: Kinetics of product formation by complex. Formation of riboflavin () and LUM () from PYR and S-DHB (A) and from R-DHB (B) is shown. C, velocities of riboflavin formation from S-DHB (solidline) and R-DHB (dashedline). The reaction mixtures contained 180 µMS-DHB or R-DHB, 800 µM PYR, and 90 µg of complex in 1 ml potassium phosphate buffer, 100 mM, pH 7.0, at 37 °C. The lines were calculated as described under ``Results.''



As shown in Fig. 8, the partitioning factor has a maximum value at a pH around 7.5. Both lumazine synthase and riboflavin synthase have their respective pH optimum in the same range.


Figure 8: pH dependence of the partitioning coefficient . The reaction mixture contained 200 µM PYR, 50 µMS-DHB and 22 µg of complex in 120 µl of potassium phosphate/Tris buffer (100 mM) at 37 °C.



The partitioning factor characterizing the flow of intermediate through the channeled and unchanneled pathway is independent of protein concentration (data not shown).


DISCUSSION

Substrate channeling in biological systems has been the focus of considerable controversy. The arguments have been summarized in a series of recent discussion papers (Ovádi, 1991).

Convincing and detailed evidence in favor of channeling has been obtained for tryptophan synthase of Escherichia coli (Creighton, 1970; Dunn et al., 1990; Anderson et al., 1991). The enzyme complex consists of an subunit catalyzing the formation of indole from indolyl glycerol phosphate and a subunit converting indole to tryptophan. The indole moiety is supposed to pass rapidly through a channel inside the dimeric protein from the active site of the subunit to the active site of the subunit. The activity of the subunit is low unless the subunit is charged with the second substrate, serine, which is required for the conversion of indole to tryptophan. As a consequence, virtually no free indole is formed, and channeling is perfect.

In other systems, results formerly attributed to channeling have been reinterpreted in terms of free diffusion kinetics on the basis of more detailed kinetic studies (Kvassman and Pettersson, 1989a, 1989b). Whereas a universally accepted definition of the criteria for channeling is not available, the absence of a lag phase for the second step in a sequential reaction is crucial.

The formation of riboflavin by the complex is best described by a leaky channel model. This is especially true when both substrates PYR and DHB are present in high concentrations. At low concentrations of the pyrimidine substrate, the partitioning factor reaches values greater than 0.8.

An important consequence of channeling is the high velocity for the conversion of ``nascent'' LUM. The velocity of riboflavin formation has its maximum value at t = 0 and remains high, while the lumazine intermediate LUM is being generated from the precursors PYR an DHB by the subunits but decreases by a factor of more than 30 after the consumption of one of the subunit substrates, despite the high concentration of the transient lumazine intermediate in the bulk solvent.

The slow rate of riboflavin formation from transient LUM (after consumption of PYR or DHB) is a direct consequence of the high apparent K of the subunits in the complex for the lumazine substrate. Thus, at a maximum concentration of 45 µM LUM in Fig. 2, the rate of riboflavin formation can be determined to be less than 5% of V. It also follows that the formation of riboflavin by channeling in the initial part of the experiment in Fig. 2proceeds at a velocity equivalent to V even at t = 0, i.e. under conditions where the bulk concentration of LUM is virtually zero. The efficiency of the subunits is thus dramatically enhanced by channeling.

The structure of the icosahedral subunit capsid has been determined at high resolution. Each of the 60 active sites is located at the interface of two adjacent subunits within the pentamer substructures of the capsid, and in close proximity of the inner capsid surface. Thus, substrates and products could easily find access to the active site from the central cavity. On the other hand, it is not clear how the exchange of substrates and products between the bulk solvent and the active site proceeds. The channels running parallel to the five-fold symmetry axes of the capsid would allow the passage of substrates PYR and DHB, but are too narrow for riboflavin and even for LUM (Ladenstein et al., 1994). Major dynamic motions of the capsid may be necessary to enable the exit of LUM or riboflavin. Under saturation conditions for the substrates, PYR and DHB, the molecule can produce about 0.7 molecules of riboflavin per second. In addition, the enzyme molecule will produce about 1.5 molecules of LUM. Local molecular motions proceed on the time scale of picoseconds. The catalytic cycle of the enzyme proceeds about 11 orders of magnitude more slowly, and this would leave ample time for major dynamic motions of the capsid as a whole, which could enable molecules as large as LUM and riboflavin to slip in and out of the protein molecule.

On the other hand, the presumptive temporary entrapment of the intermediate LUM inside the capsid where it is generated could provide a straightforward explanation of the unusual kinetic properties of the enzyme complex. The molecule temporarily trapped inside the capsid would have time to find the active site of the subunits where it is converted to riboflavin. The increase of K by a factor of 10, which accompanies the enclosure of the subunit trimer in the subunit capsid, could be a consequence of hampered diffusion of LUM through the capsid wall.

The pyrimidine PYR is both a product of the subunit and a substrate of the subunit. Thus, the intermediate PYR could enhance the overall velocity by channeling from the subunits to the subunits, i.e. in the opposite direction as LUM. However, no experimental evidence for this plausible mechanism has been obtained up to now.

In summary, the complex is a very efficient device for the rapid formation of riboflavin at low concentration of PYR and DHB. This raises the question how an enzyme complex with these remarkable properties could have developed. Factors providing the necessary selective pressure for the evolution of the complex could have been (i) a requirement to produce riboflavin at a high rate with a minimum amount of catalyst or (ii) a requirement to avoid the accumulation of unstable intermediates which might be lost by wasteful side reactions.

The chemical stability of the intermediates PYR and DHB is limited. Thus, DHB can decompose spontaneously under formation of diacetyl, and the reaction is accelerated by the presence of acids and bases. The diaminopyrimidine PYR is sensitive to molecular oxygen and can decompose under formation of a tricyclic pyrimidopteridine (Cresswell et al., 1960). Moreover, the isolation of 6-methyl-7-dihydroxyethyl-8-ribityllumazine from the culture fluid of riboflavin-deficient B. subtilis mutant strains (Bresler et al., 1976) suggests that PYR can react with carbohydrates in bacterial cells.

The riboflavin requirement of B. subtilis under laboratory culture conditions is low. Riboflavin mutants reach their maximum growth rate at a concentration of about 50 µg of riboflavin/liter. Nevertheless, B. subtilis has developed a complex regulatory system for the riboflavin pathway, which allows up-regulation of expression of biosynthetic enzymes by a factor of at least 30 from the basic level (Bacher and Mailänder, 1978). Under laboratory conditions, B. subtilis wild strains are continually in a state of maximum repression of the riboflavin pathway. The potential for up-regulation becomes manifest only in riboflavin-starved riboflavin mutants. Under conditions of maximum derepression, the lumazine synthase/riboflavin synthase complex accounts for as much as about 0.5% of cellular protein. This regulatory potential should enable the microorganism to produce riboflavin at a level by far exceeding its basic metabolic requirement.

The evolution of this regulatory system could reflect a need for massive riboflavin overproduction in natural habitats of B. subtilis. The selective pressure that was responsible for the evolution of the regulatory system could also have worked to streamline the metabolic process by the device of substrate channeling.

Our understanding of metabolic and regulatory aspects of riboflavin biosynthesis in B. subtilis is still incomplete. A more detailed understanding of channeling in the lumazine synthase/riboflavin synthase will require the determination of the individual rate constants in the kinetic network shown in Fig. 5.

  
Table: Steady state kinetic parameters of the proteins under study (data from Bacher et al., 1980, and from Kis et al., 1985)

V, V, maximum velocities of lumazine synthase () and riboflavin synthase (), respectively. K, K, K, Michaelis constants for the substrates indicated.


  
Table: Kinetic simulation of riboflavin formation by the / mixture

The refined values were obtained by numerical iteration and yield the line curves in Fig. 4. V and V, maximum velocities of lumazine synthase () and riboflavin synthase (), respectively. K, K, Michaelis constants for the substrates indicated.


  
Table: Kinetic simulation of riboflavin formation by the complex

The refined values were obtained by numerical iteration and yield the line curves in Fig. 2. V, V, maximum velocities of lumazine synthase () and riboflavin synthase (), respectively. K, K, Michaelis constants for the substrates indicated.



FOOTNOTES

*
This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie and Grant EC ERBCHRXCT 930166 from the European Community. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to Professor Ivar Ugi on the occasion of his 65th birthday.

The abbreviations used are: DHB, 3,4-dihydroxy-2-butanone 4-phosphate; LUM, 6,7-dimethyl-8-ribityllumazine; PYR, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione; RIB, riboflavin.

M. Fischer, K. Kugelbrey, and A. Bacher, unpublished data.

J. Scheuring, M. Fischer, M. Cushman, J. Lee, A. Bacher, and H. Oschkinat, submitted for publication.


ACKNOWLEDGEMENTS

We thank Angelika Kohnle and Cornelia Krieger for skillful technical assistance and for help with the preparation of the manuscript. We are grateful to J. Rudolph and J. Stubbe for sharing of unpublished data.


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