Mechanism of the Polymerization Reaction Initiated and Catalyzed by

Polyhydroxybutyrate (PHB) synthases (polymerases) catalyze the polymerization of the coenzyme A thioester of 3-hydroxybutyrate to PHB. The Ralstonia ...
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Biomacromolecules 2003, 4, 504-509

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Mechanism of the Polymerization Reaction Initiated and Catalyzed by the Polyhydroxybutyrate Synthase of Ralstonia eutropha Shiming Zhang,† Steve Kolvek,† Robert W. Lenz,‡ and Steve Goodwin*,† Department of Microbiology and Department of Polymer Science and Engineering, University of Massachusetts, Amherst, Massachusetts 01003 Received August 30, 2002; Revised Manuscript Received January 8, 2003

Polyhydroxybutyrate (PHB) synthases (polymerases) catalyze the polymerization of the coenzyme A thioester of 3-hydroxybutyrate to PHB. The Ralstonia eutropha PHB synthase purified from recombinant E. coli cells exists in aqueous solution in both monomeric (single subunit) and homodimeric (two subunits) forms in equilibrium. Several lines of evidence suggest that the homodimer is the active form of the synthase. The initial mechanistic model for the polymerization reaction proposed that two different thiol groups form the catalytic site. The cysteine at 319 has been shown to provide one thiol group that is involved in the covalent catalysis, but a second thiol group on the same protein molecule has not yet been identified. It is suggested that cysteines at 319 from each of the two molecules of a homodimer synthase provide two identical thiol groups to jointly form a single catalytic site. To verify this model using the strategy of in vitro reconstitution, heterodimers composed of the wild-type subunit and of the C319 mutated subunit were constructed and the activities at various ratios of the wild-type subunit to the mutated subunit were measured. The experimental results indicate that the homodimer is the active form of the enzyme, that the heterodimer containing the mutated subunit has no activity, and that a single cysteine is not sufficient for catalysis. Two identical thiol groups from C319 residues on each subunit of the homodimer are required to form the catalytic site for the initiation and propagation reactions. We further demonstrate that a dimer synthase that has initiated the polymerization reaction (primed synthase) is significantly more stable against dissociation than the unprimed (unreacted) dimer synthase. These two properties explain the nature of lag phenomenon during the in vitro polymerization reaction catalyzed by this enzyme Introduction Polyhydroxyalkanoates (PHAs) are synthesized by a wide range of bacteria as carbon and energy storage products. PHA synthases are polymerases that are the key enzymes catalyzing the polymerization of CoA derivatives of hydroxyalkanoic acids (HACoA). Three types of PHA synthases have been proposed based on their substrate specificity and enzyme structure. Type I and type III synthases both preferentially polymerize short chain hydroxyalkanoic acid monomers (HACoAs) containing 3-5 carbon atoms.1,2 However, type I synthases are composed of only a single subunit (a single protein molecule), PhaC, whereas type III synthases are composed of two different subunits, PhaC and PhaE.1,3-6 Type II synthases are again composed of a single subunit, PhaC, but preferentially polymerize HACoAs containing more than 5 carbon atoms.7-9 The PHB synthase from Ralstonia eutropha (PhaCRe) is the representative of the type I synthases that has been the most extensively studied of the PHA synthases.1,6,10-15 PhaCRe exists in aqueous solution as an equilibrium between * To whom correspondence should be addressed. Phone: 413-545-4604. Fax: 413-545-1578. E-mail: [email protected]. † Department of Microbiology. ‡ Department of Polymer Science and Engineering.

monomeric and homodimeric enzyme molecules.6,11 Our previous studies show that synthase activity is reversibly lost during dilution and that the dimer has a significantly higher specific activity than the monomer.11 Furthermore, the observed relationship between PHB molecular weights and substrate-to-enzyme ratios is consistent with a single polymer chain being formed by the homodimer synthase, which again implies that dimer is the active form of the enzyme.11 When purified PhaCRe is used for the in vitro polymerization of HACoA monomers, a lag period of significant but varied lengths occurs before the reaction reaches a maximum rate.6,10 The lag period can be eliminated either by reacting the enzyme with substrate (priming reaction) 6,13 or by adding multihydroxyl compounds to the enzyme solution, which promotes the conversion of monomer to dimer.11 The dissociation of dimer to monomer is increased by diluting the enzyme, which causes a reversible loss of the enzyme activity.11 The original mechanistic model of the PHA synthase catalyzed polymerization reaction proposed that two different thiol groups participate in the covalent catalysis.16,17 For PhaCRe, one such thiol group has been conclusively demonstrated to be provided by the cysteine at position 319 (C319).6,13,18 However, attempts to identify a second thiol group on the same subunit have not been successful. It should

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also be noted that the only cysteine shown to be highly conserved across 30 PHA synthases was C319. This observation led to the suggestion that the second thiol group might be provided by the other subunit in the homodimer synthase.19 On the basis of our observation that the homodimer synthase appears to produce a single polymer chain, we have proposed that the two essential thiol groups are provided by the C319 on each subunit of the homodimer and that these two C319 form a single catalytic site.11 Because the two thiol groups are identical, they could alternatively function as the site for accepting substrate and the site for covalently binding the growing polymer chain. According to this mechanism, the polymer chain would transfer back and forth, covalently, between the two thiol groups for each propagation step. This model predicts that homodimer is the only active form of the synthase and that a single cysteine is not sufficient for catalysis. To verify that the homodimer is the active form of the synthase and that a single cysteine is not sufficient for catalysis, we examined the activity of a heterodimer that was reconstituted in vitro from wild-type synthase subunits and C319-mutated synthase subunits. The results demonstrate that the heterodimer has no activity and that a single cysteine is not sufficient for catalysis. The kinetic properties of dissociation of the dimeric synthase were further investigated to better understand the lag period. The results indicate that the unreacted dimeric synthase (unprimed) dissociates much faster than the dimeric synthase that is covalently bonded to a growing polymer chain after having initiated the polymerization (primed). The dramatically increased stability of the primed dimeric synthase promotes conversion of the synthase from monomer to dimer and continuously shifts the equilibrium during the polymerization reaction. Materials and Methods Mutated Synthase. Mutagenesis to change C319 to A319 was performed on plasmid pKAS46 by the recombinant PCR method,20 using four primers with the following sequences: P1:5′-CTGACACGCGGCAAGATCTCGCAG-3′ P2:5′-CCGCCCACGCCGAAGCCGAGCACGTTGATCTTG-3′

P3:5′-GCTCGGCTTCGGCGTGGGCGGCACCATTGTCTC-3′ P4:5′-CGTGGCCTCGCGCAACTGCACATG-3′

The base change in primers 2 and 3 are indicated by bold type. After recombinant PCR, the amplified fragments containing the mutation were treated with AatII and BglII and used to replace the corresponding wild-type region in the plasmid pKAS4. The mutation of TGC to GCC was confirmed by sequencing. The plasmid DNA was introduced into the E. coli strain UT5600 by the calcium chloride method.21 Isolation of PHB Synthase and Activity Assay. The recombinant E. coli strain UT5600 harboring the plasmids containing either the wild-type or the mutated synthase genes was used for synthase production. Cell culturing, harvesting,

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and breakage were performed as described by Gerngross et al.6 The purification of the PHB synthase was done using a hydrophobic interaction Methyl Econo column, (Bio-Rad Corp.) as described by Song et al.22 The purity of the synthase samples was determined by densitometric analysis on SDSPAGE gel (4-20%, Bio-Rad) after staining with Coomassie Brilliant Blue. Protein concentration was determined using the method of Bradford.23 Synthase activity was assayed spectrophotometrically by measuring the absorbance change at 236 nm because of the hydrolysis of the thioester bonds in [R]-3-hydroxybutyrylCoA (R-3HBCoA) according to the method of Fukui et al.24 One unit of activity represents the turnover of one micromole of substrate in one minute. Unless otherwise specified, the reaction was performed in 1 mL of total volume with 20 mM phosphate buffer solution at pH 7.0. Reconstitution of Synthase Dimer. It is known that purified synthase preparations contain primarily monomer and that addition of fructose can promote rapid dimerization.11,22 This method was used for the rapid in vitro reconstitution of the synthase dimer. For the initial reconstitution experiment, the synthase preparations were adjusted to equal protein concentrations of 200 µg/mL. Four types of samples were prepared: wildtype synthase, wild-type and mutated synthase, wild-type synthase plus fructose, and wild-type and mutated synthase plus fructose. For the samples containing both the wild-type and the mutated synthase, equal volumes of the wild-type synthase and the mutated synthase were mixed. To produce the samples containing fructose, solid fructose was added to a final concentration of 70% (w/v). After fructose was completely dissolved, the samples were allowed to stand for 40 min. To have equal amounts of wild-type enzyme in all samples, activity measurement were performed with 20 µL of the wild-type synthase samples and 40 µL of the mixture of wild-type and mutated synthase samples. In a more detailed quantitative analysis, after adjusting the synthase preparations to equal protein concentrations of 200 µg/mL, the wild-type and the mutated synthase samples were mixed together at varying volume ratios in the absence of fructose to give a total volume of 100 µL. After standing for 10 min, 100 µL of fructose solution (1 g of fructose dissolved in 1 mL of reaction buffer) was added. The mixture was left at room temperature for another 40 min before 50 µL of the mixture was taken for activity assay. Priming Synthase. The priming reaction was carried out at room temperature using an equivalent of 2 µg of synthase in a 70% aqueous solution of fructose (w/v) with 35 nmol of [R]-3HBCoA in a total volume of 20 µL for 10 min. Dilution Experiment. A dilution method was employed to examine the stability of the primed and unprimed synthase homodimer. Synthase samples of 200 µL were amended with fructose to a final concentration of 70%(w/v) to promote the formation of the dimer. The sample was then divided into two aliquots of 100 µL each. One aliquot was reacted with a specific amount of [R]-3HBCoA (500 nmol, in 70% fructose (w/v)) for 10 min. The second aliquot was amended with the same volume of 70% fructose solution. From these two preparations, 20 µL of each were taken and added to

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Figure 2. Effect of mutated synthase subunits (A319) on the initial activity of the primed wild-type homodimer synthase. (b) Initial activity of wild-type homodimer in the absence of mutated subunits; (2) initial activity of wild-type homodimer in the presence of mutated subunits. Figure 1. Rate of polymerization for wild-type homodimer and mutated homodimer. (9) Wild-type homodimer (C319); (×) mutated homodimer (A319).

980 µL of the reaction buffer and held for various periods of incubation before 10 µL of [R]-3HBCoA (35 nmoles) was added to measure the residual activity of the synthase. The initial activity was obtained by adding the synthase sample (20 µL) to 990 µL of reaction buffer containing 35 nmol of [R]-3HBCoA. R-3-Hydroxybutyryl CoA. This compound was a kind gift from Dr. David P. Martin (Metabolix Inc. MA).25 Results Construction and Properties of Mutated Synthase. The mutated A319 synthase was constructed for in vitro reconstitution with the wild-type synthase. The mutated A319 synthase was previously demonstrated to have no polymerization activity, but it had the same conformation as the wildtype synthase.6 In the present study, the mutated A319 synthase and the wild-type synthase were expressed in the same strain of E. coli and were purified under the same conditions. The mutated A319 synthase was found to behave the same as the wild-type synthase during the purification steps. Its preparation showed similar yield and purity as the wild-type synthase. Nondenatured PAGE results also showed a similar pattern between the A319 mutated and the wildtype synthase preparations (data not shown) indicating that they have a similar equilibrium constant between monomer and dimer. Heterodimer Synthase Activity. The basic question is whether a single subunit of the wild-type synthase is sufficient for polymerization activity. To answer this question experimentally, it is first necessary to demonstrate that the mutated homodimer synthase does not have polymerization activity and that the presence of mutated synthase does not inhibit the polymerase activity of wild-type homodimer. Figure 1 is a plot of monomer conversion as a function of reaction time for both the wild-type synthase and the mutated synthase. The earlier report6 that the mutated A319 synthase did not have detectable polymerization activity is confirmed by the results in this plot. The activity of the wild-type synthase during the polymerization of 3HBCoA was examined in the absence and in the presence of the mutated synthase. In this case, a preparation of primed, wild-type homodimer was mixed with

Table 1. Initial Activity of Wild-Type and Mutated Synthase treatment

initial activity (µmol/min)

wild-type synthase wild-type synthase and mutated synthase wild-type synthase with fructose wild-type synthase and mutated synthase with fructose

0.001 0.001 0.084 0.044

an equal amount of mutated synthase. It was found that the presence of the mutated synthase did not change the initial rate of the polymerization reaction as shown in Figure 2. This result confirmed that the mutated synthase does not inhibit the polymerization reaction initiated and catalyzed by the primed synthase. However, with the unprimed wildtype synthase, after the initial reaction, there is a decrease in the rate at which the activity accelerates which is proportional to the relative amount of the mutated subunit present (data not shown). This result suggests that the mutated synthase does not inhibit the catalytic reaction directly, but it may influence the formation of the wild-type homodimer. It should also be noted that, as discussed above, the homodimer can exist in two states, either in the less reactive unprimed state or in the primed state, which is formed after the enzyme has initiated a polymerization reaction and which has a much higher reactivity. We further investigated whether the heterodimer (C319/ A319) had polymerase activity by varying the proportions of mutated and wild-type subunits in reconstitution experiments. First, we measured the initial activity of the wild-type synthase (C319) alone and then in an equimolar ratio of wildtype synthase and the mutated synthase (C319 and A319) in the absence of fructose (see next paragraph). The total amount of wild-type synthase was the same in both reactions. As presented in Table 1, both reactions had very low activities as would be expected in the absence of fructose, because most of the synthase molecules were in the monomeric form in the absence of fructose. Addition of multihydroxyl compounds, such as fructose, to the synthase solution increases the enzyme activity by promoting the conversion of monomer to dimer.11 Therefore, we examined the change in activity in the two reactions discussed above after the addition of fructose. One reaction contained only the wild-type synthase, and the other contained the same amount of the wild-type synthase and an additional equal amount of mutated synthase. For the wild-

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Figure 3. Initial activities of the reconstituted synthase samples containing different molar ratios of the wild-type synthase (C319) and the mutated synthase (A319) subunits. (2) Calculated values assuming C319/A319 heterodimer has 50% of the activity of the wild-type dimmer; (b) calculated values assuming C319/A319 heterodimer has no activity; (9) experimental results.

type synthase alone, the addition of fructose caused an approximate increase in the activity of 80-fold, indicating that a large amount of dimeric synthase was formed after the addition of fructose. In contrast, the increased activity for the treatment that also contained mutated synthase was closer to 40-fold. This result is a clear indication that the wild-type synthase is affected by the formation of heterodimers with the mutated synthase. On the average, onehalf of the wild-type subunits can be expected to form homodimers with other wild-type subunits, whereas one-half of the wild-type subunits can be expected to form heterodimers with the mutated subunits. The sample containing both wild-type and mutated synthase had only 52% as much activity as the sample with only wild-type synthase, suggesting that the wild-type subunits in heterodimers have no activity. The investigation of heterodimer (C319/A319) polymerase activity was extended by varying the proportions of mutated and wild-type subunits in reconstitution experiments. Addition of fructose to the mixtures would form three types of dimers: C319/A319, C319/C319, and A319/A319. In equilibrium, the three types of dimeric synthases formed should be present in the amounts determined by the following equation: ( fC319) 2 + 2 fC319 fA319 + ( fA319)2 ) 1

Figure 4. A. Activity loss of the unprimed and the primed synthases after dilution. B. Logarithm of the relative residual activity as a function of time after dilution. (A0 and At are the enzyme activities before and during the incubation time t (seconds), respectively.) (b) Unprimed synthase; (2) primed synthase. C. Expanded plot of the first 40 s of Figure 4B for the unprimed synthase.

where f is defined as the fraction of each type of synthase and fC319 + fA319 ) 1. If a single cysteine was sufficient for catalysis, the activity in a heterodimer (C319 /A 319) should be one-half of that in the wild-type dimer; otherwise, it should have no activity. For these two possibilities, the calculated and observed initial activities as a function of the percentage of the wild-type synthase in the sample are shown in Figure 3. If the heterodimer was active, the initial activity would decrease in direct proportion to the decrease in relative concentration of the wild-type synthase. If the heterodimer had no activity, a plot of the initial activity as a function of the relative concentration of the wild-type synthase should be concaved upward. The experimental results shown in Figure 3 confirm that the type of plot obtained fits well with the calculated values which result from assuming that the heterodimer has no activity.

Dissociation Kinetics. A slow rate of conversion of monomeric to dimeric forms can explain, in part, the lag period observed for these in vitro polymerization reactions,11 but it is not known what factor shifts the equilibrium toward dimer formation to maximize the enzyme’s specific activity. The dimeric synthase has two states, one of which is the unprimed state before the polymerization reaction is initiated by the enzyme and the other is the primed state after the polymerization reaction has been initiated. The dissociation kinetics were investigated for both of these states by employing a dilution method as described in the Materials and Method section. Figure 4A shows the loss of the initial activity, which is the activity of the synthase before dilution, with incubation time after dilution. It was found that the unprimed synthase lost 70% of its activity within 40 s after dilution and 87% of its activity within 10 min. In contrast,

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the primed synthase lost only 29% of its activity within 10 min and only 31% of its activity within 20 min. After a 50-fold dilution, the original equilibrium between dimer and monomer is considerably shifted toward the monomer and the dissociation of the synthase from dimer to monomer should occur fairly rapidly after dilution. During this period, the rate of decrease in dimer concentration can be determined from the following equation: d[Ed]t /dt ) -k-1[Ed]t + k1[Em]t where [Ed]t is the dimer concentration and [Em]t the monomer concentration at time t and k-1 and k1 are the dissociation and association rate constants. The second term on the right for monmer association is negligible, as discussed below, so the concentration of the dimer at time t can therefore be expressed as ln([Ed]t/[Ed]0) ) -k-1t where [Ed]0 is the initial concentration of the dimer. Because the activity of PHB synthase is directly determined by the dimer concentration, ref 11 and this study, this relationship can also be expressed as ln(At /A0) ) -k-1t where A0 and At are the activities of the synthase at time zero and at time t of the incubation, respectively. If a plot of this equation has an initial linear region, the contribution from the monmer association is negligible and can be ignored, so that data for this region can be used to calculate the dissociation rate constant. The initial regions in Figure 4B for both the primed and unprimed synthases were linear (data not shown), so the values of k-1 could be obtained from this experiment. A plot of ln(At/A0) as a function of time is shown in Figure 4B. For the unprimed synthase, a rapid activity loss was detected within the first 40 s of the incubation, as shown in Figure 4C, which was followed by a gradually slower process of activity loss. Using the data for the first 40 s, the k-1 value for the unprimed synthase was estimated to be 0.026 s-1. The data for the primed synthase is not as complete, but a plot of the first 120 s for the primed synthase gives an estimated value of 0.000 95 s-1, which is 27 times smaller than that of the unprimed synthase. At the end of the incubation period, an equilibrium is established, and the concentration of the dimer synthase can be estimated from the residual activity compared with the initial activity. The expression for the dissociation constant is as follows: Kd ) [Em]2/[Ed] where [Em] and [Ed] are the concentrations of the monomer and the dimer of the synthase, respectively. From the data obtained, the dissociation constants Kd for the unprimed synthase and the primed synthase were estimated to be 350 and 5.3 nM, respectively. The rate constants for dimerization (k1) for unprimed and primed synthases were estimated to be 74 and 180 mM-1 s-1, respectively, based on Kd ) k-1/

Zhang et al.

k1. These data show that primed dimer synthase is much more stable than the unprimed synthase, primarily because of the much slower dissociation rate of the primed dimer synthase. Discussion A key question about the polymerization reaction mechanism for the PHB synthase, which was addressed in this work, is whether the active center requires a second thiol group. If a second thiol group is required, then additional questions are where is the location of the second thiol group and how does it coordinate with the first in catalyzing the propagation reaction. It is suggested that a single polymer chain is produced by a homodimer synthase as indicated by the relationship between polymer molecular weight and the substrate-to-enzyme molar ratio.11 This relationship requires that a homodimer synthase contains only a single catalytic site. Because a homodimer contains two identical thiol groups on the C319 of the two subunits, it can be suggested that these two thiol groups jointly form the catalytic site, which binds covalently to both the growing polymer chain and the incoming monomer. In this case, a single cysteine would not be sufficient for initiation and catalysis, and a heterodimer consisting of one wild-type subunit and one C319 mutated synthase subunits, such as the A319 mutated synthase studied here, would have no activity. In this study, the above prediction was tested by examining the activity in the heterodimer synthase containing both wildtype and the mutated synthase subunits. The mutated A319 synthase has previously been demonstrated to be inactive but to have essentially the same conformation as the wildtype synthase.6 In addition, we observed that the A319 dimer has a similar dissociation tendency as the wild-type dimer synthase, as indicated by its nondenatured PAGE pattern (data not shown). Therefore, dimerization of the mixture of wild-type and the mutated synthase would be expected to be similar to the dimerization of wild-type synthase alone. Only 52% of the activity of the wild-type homodimer was detected in the sample consisting of equal amounts of wildtype and A319 mutated synthases compared with that of the same amount of wild-type synthase after the dimerization was promoted by addition of fructose in each case. This result is in very good agreement with the calculated value of 50%, which assumes that there is both the same ability for the formation of heterodimer and the homodimer and that the heterodimer has no activity. The results from a more detailed quantitative activity analysis on a series of samples consisting of the wild-type and the mutated synthases in various proportions provided further supportive evidence that the heterodimer has no activity. Taken together, these results indicate that the polymerization reaction requires two identical thiol groups that are provided by identical cysteines, one on each subunit of the homodimeric synthase. The need for dimer formation alone is not sufficient to fully account for the lag phase in the in vitro polymerization reaction. To obtain the activity increase observed during the polymerizaton reaction, it can be postulated that either polymer formation stimulates the synthase activity25 or that increasing amounts of dimer are formed during polymeri-

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zation reaction. Our previous studies, which showed that the synthase sample in 70% (w/v) fructose did not have a lag phase,11 indicated that polymer formation is not necessary for stimulating synthase activity. The results of the present study clearly indicate that the primed dimeric synthase is much more stable than the unprimed dimeric synthase against dissociation into the inactive monomeric form. Once the polymerization reaction has begun, the equilibrium between the monomer and the dimer is continuously shifted toward the dimer. The higher stability of the primed synthase dimer could be due either to a conformational change in the enzyme resulting in a stronger mutual interaction between two subunits or to the association of the polymer chain with the hydrophobic surfaces of both enzyme subunits. The high stability of the primed dimeric synthase could be an important in vivo regulatory mechanism for controlling carbon storage in R. eutropha because the synthase is constitutively expressed in this bacterium, but PHB is only produced and stored under conditions of carbon excess. Acknowledgment. This work was supported by a grant to R.W.L. and S.G from the New Energy and Industrial Technology Development Organization, Japan. References and Notes (1) Liebergesell, M.; Sonomoto, K.; Madkour, M.; Mayer, F.; Steinbu¨chel, A. Eur. J. Biochem. 1994, 226, 71-80. (2) Haywood, G. W.; Anderson, A. J.; Dawes, E. A. Biotechnol. Lett. 1989, 11, 471-476. (3) Slater, S. C.; Voige, W. H.; Dennis, D. E. J. Bacteriol. 1988, 170, 4431-4436. (4) Liebergesell, M.; Steinbu¨chel, A. Eur. J. Biochem. 1992, 209, 135150. (5) Peoples, O. P.; Sinskey, A. J. J. Biol. Chem. 1989, 264, 1529815303. (6) Gerngross, T. U.; Snell, K. D.; Peoples, O. P.; Sinskey, A. J.; Csuhai, E.; Masamune, S.; Stubbe, J. Biochemistry 1994, 33, 9311-9320.

Biomacromolecules, Vol. 4, No. 3, 2003 509 (7) Ren; de Roo, G.; Kessler, B.; Witholt, B. Biochem. J. 2000, 349, 599-604. (8) Kraak, M. N.; Smits, T. H. M.; Kessler, B.; Witholt, B. J. Bacteriol. 1997, 179, 4985-4991. (9) Huisman, G. W.; Wonink, E.; Meima, R.; Kazemier, B.; Terpstra, P.; Witholt, B. J. Biol. Chem. 1991, 266, 2191-2198. (10) Williams, M. D.; Fieno, A. M.; Grant, R. A.; Sherman, D. H. Prot. Expr. Purific. 1996, 7, 203-211. (11) Zhang, S.; Yasuo, T.; Lenz, R. W.; Goodwin, S. Biomacromolecules 2000, 1, 244-251. (12) Jia, Y.; Kappock, J.; Frick, T.; Sinskey, A. J.; Stubbe, J. Biochemistry 2000, 39, 3927-3936. (13) Wodzinska, J.; Snell, K. D.; Rhomberg, A.; Sinkey, A. J.; Biemann, K.; Stubbe, J. J. Am. Chem. Soc. 1996, 118, 6319-6320. (14) Haywood, G. W.; Anderson, A. J.; Dawes, E. A. FEMS Microbiol. Lett. 1989, 57, 1-6. (15) Mu¨h, U.; Sinskey, A. J.; Kirby, D. P.; Lane, W. S.; Stubbe, J. Biochemistry 1999, 38, 826-837. (16) Doi, Y.; Kawaguchi, Y.; Koyama, N.; Nakamura, S.; Hiramitsu, M.; Yoshida, Y.; Kimura, H. FEMS Microbiol. ReV. 1992, 103, 103108. (17) Ballard, D. G. H.; Holmes, P. A.; Senior, P. J. In Recent adVances in mechanistic and synthesis aspects of polymerization; Fontanille, M., Guyot, A., Eds.; Reidel (Kluwer): Lancaster, U.K., 1987; Vol. 215, pp 293-314. (18) Jia, Y.; Yuan, W.; Wodzinska, J.; Park, C.; Sinskey, A. J.; Stubbe, J. Biochemistry 2001, 40, 1011-1019. (19) Rehm, B. H. A.; Steinbu¨chel, A. Int. J. Biol. Macromol. 1999, 25, 3-19. (20) Higuchi, R. In PCR Protocols; Innis, M. A., Gelfand, D. H., Sninsky, J. J., White, T. J., Eds.; Academic Press: San Diego, CA, 1990; pp 177-183. (21) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning: a Laboratory Manual; Cold Spring Harbor Laboratory Press: Plainview, NY, 1989. (22) Song, J. J.; Zhang, S.; Lenz, R. W.; Goodwin, S. Biomacromolecules 2000, 1, 433-439. (23) Bradford, M. M. Anal. Biochem. 1976, 72, 248-254. (24) Fukui, T.; Yoshimoto, A.; Matsumoto, M.; Hosokawa, S.; Saito, T.; Nishikawa, H.; Tomita, K. Arch. Microbiol. 1976, 110, 149156. (25) Gerngross, T. U.; Martin, D. P. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 6279-6283.

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