Tài liệu Báo cáo khoa học: Mechanism of dihydroneopterin aldolase NMR, equilibrium and transient kinetic studies of the Staphylococcus aureus and Escherichia coli enzymes docx

Mechanism of dihydroneopterin aldolase NMR, equilibrium and transient kinetic studies of the Staphylococcus aureus and Escherichia coli enzymes Yi Wang, Yue Li, Yan Wu and Honggao Yan Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA Dihydroneopterin aldolase (DHNA, EC 4.1.2.25) catalyzes the conversion of 7,8-dihydro-d-neopterin (DHNP) into 6-hydroxymethyl-7,8-dihydropterin (HP) in the folate biosynthetic pathway, one of the principal targets for developing antimicrobial agents [1]. Folate cofactors are essential for life [2]. Most micro-organ- isms must synthesize folates de novo. In contrast, mam- mals cannot synthesize folates because of the lack of three enzymes in the middle of the folate pathway, and they therefore obtain folates from the diet. DHNA is the first of the three enzymes that are absent in mam- mals and therefore an attractive target for developing antimicrobial agents [3]. DHNA is a unique aldolase in two respects. First, it requires neither the formation of a Schiff’s base between the substrate and enzyme nor metal ions for catalysis [4]. Aldolases can be divided into two classes based on their catalytic mechanisms [5,6]. Class I aldo- lases require the formation of a Schiff’s base between an amino group of the enzyme and the carbonyl of the substrate, whereas class II aldolases require a Zn 2+ ion at their active sites for catalysis. The proposed catalytic mechanism for DHNA [4,7,8] is similar to that of class I aldolases, but the Schiff’s base is embedded in the substrate (Fig. 1). Secondly, in addition to the aldo- lase reaction, DHNA also catalyzes the epimerization Keywords dihydroneopterin aldolase; Escherichia coli; folate biosynthesis; mechanism; Staphylococcus aureus Correspondence H. Yan, Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA Fax: +1 517 353 9334 Tel: +1 517 353 5282 E-mail: yanh@msu.edu Website: http://www.bch.msu.edu/faculty/ yan.htm *These authors have contributed equally to this work (Received 13 January 2007, revised 14 February 2007, accepted 28 February 2007) doi:10.1111/j.1742-4658.2007.05761.x Dihydroneopterin aldolase (DHNA) catalyzes both the cleavage of 7,8-dihydro-d-neopterin (DHNP) to form 6-hydroxymethyl-7,8-dihydro- pterin (HP) and glycolaldehyde and the epimerization of DHNP to form 7,8-dihydro-l-monapterin (DHMP). Whether the epimerization reaction uses the same reaction intermediate as the aldol reaction or the deprotona- tion and reprotonation of C2¢ of DHNP has been investigated by NMR analysis of the reaction products in a D 2 O solvent. No deuteration of C2¢ was observed for the newly formed DHMP. This result strongly suggests that the epimerization reaction uses the same reaction intermediate as the aldol reaction. In contrast with an earlier observation, the DHNA- catalyzed reaction is reversible, which also supports a nonstereospecific retroaldol ⁄ aldol mechanism for the epimerization reaction. The binding and catalytic properties of DHNAs from both Staphylococcus aureus (SaDHNA) and Escherichia coli (EcDHNA) were determined by equilib- rium binding and transient kinetic studies. A complete set of kinetic con- stants for both the aldol and epimerization reactions according to a unified kinetic mechanism was determined for both SaDHNA and EcDHNA. The results show that the two enzymes have significantly different binding and catalytic properties, in accordance with the significant sequence differences between them. Abbreviations DHMP, 7,8-dihydro-L-monapterin; DHNA, dihydroneopterin aldolase; DHNP, 7,8-dihydro- D-neopterin; EcDHNA, E. coli dihydroneopterin aldolase; GA, glycolaldehyde; HP, 6-hydroxymethyl-7,8-dihydropterin; HPO, 6-hydroxymethylpterin; MP, L-monapterin; NP, D-neopterin; SaDHNA, S. aureus dihydroneopterin aldolase. 2240 FEBS Journal 274 (2007) 2240–2252 ª 2007 The Authors Journal compilation ª 2007 FEBS at C2¢ of DHNP to generate 7,8-dihydro-l-monapterin (DHMP) [7], but the biological function of the epi- merase reaction is not known at present. The aldolase and epimerase reactions are believed to involve a com- mon intermediate as shown in Fig. 1 [4,7,8]. Both reac- tions involve the retroaldol cleavage of the C–C bond between C1¢ and C2¢. Epimerization results from the re-formation of the C–C bond after the reorientation of glycolaldehyde, which exposes the opposite face of the aldehyde. The mechanism of the epimerization reaction is very similar to that catalyzed by l-ribulose- 5-phosphate 4-epimerase [9], which also follows aldol chemistry [10], but the two enzymes are different in structure and have no apparent sequence identity. l-Ribulose-5-phosphate 4-epimerase has 26% identity with the class II l-fuculose-1-phosphate aldolase and requires a Zn 2+ ion for catalysis [9]. DHNA is unique because it catalyzes both aldolase and epimerase reac- tions, whereas l-ribulose-5-phosphate 4-epimerase and l-fuculose-1-phosphate aldolase catalyze only one type of reaction. Interestingly, DHNAs from Gram-positive and Gram-negative bacteria have some unique sequence motifs. Figure 2 shows the amino-acid sequence align- ment of DHNAs from 11 bacteria. The first five enzymes are from Gram-positive bacteria, and the rest are from Gram-negative bacteria. The identities between enzymes from Gram-positive bacteria range from 39% to 45% and those between Gram-negative bacteria are 49–91%, but the identities between Gram-positive and Gram- negative bacterial enzymes are < 30%. Many differ- ences between enzymes from Gram-positive and Gram- negative bacteria are at or near their active centers [8]. DHNA was first identified in Escherichia coli (EcDHNA) by Mathis and Brown in 1970 [4]. There were few studies on DHNA until 1998, when Hennig and coworkers determined the crystal structures of DHNA from Staphylococcus aureus (SaDHNA) and its complex with the product HP [8]. In the same year, Haussmann and coworkers demonstrated that the enzyme has both aldolase and epimerase activities and determined the steady-state kinetic parameters for both reactions [7]. In 2000, the Wu ¨ thrich group pub- lished the total sequential resonance assignment of the 110-kDa homo-octomeric SaDHNA [11], which was a model system for the development of TROSY (transverse relaxation optimized spectroscopy) NMR [12–14]. Also in 2000, Deng and coworkers measured the pK a of N5 of SaDHNA-bound 7,8-dihydrobio- pterin by Raman spectroscopy [15]. In 2002, Illarionova and coworkers showed that the protonation of the reac- tion intermediate prefers the pro-S position [16]. We are interested in understanding the catalytic mechanism of DHNA and the biochemical conse- quences of the significant sequence differences des- cribed above. Most recently, we studied the dynamic properties of apo-SaDHNA and the product complex SaDHNA–HP by molecular dynamics simulations [17] and began to investigate the functional roles of the act- ive-site residues by site-directed mutagenesis [18]. In this paper, we address the issue of whether the epim- erase reaction follows a nonstereospecific retroaldol ⁄ Fig. 1. Proposed catalytic mechanism for the DHNA-catalyzed reactions. Both aldolase and epimerase reactions follow the same reaction intermediate generated by the clea- vage of the bond between 1¢ and 2¢ carbons of the substrate. The epimerization product is generated by the re-formation of the C–C bond after the reorientation of GA, which exposes the opposite face of the aldehyde. Y. Wang et al. Mechanism and kinetics of dihydroneopterin aldolase FEBS Journal 274 (2007) 2240–2252 ª 2007 The Authors Journal compilation ª 2007 FEBS 2241 aldol mechanism [7] or an alternative mechanism via the deprotonation and re-protonation of C2¢ and report a comprehensive equilibrium and kinetic study of SaDHNA and EcDHNA, which represent DHNAs from Gram-positive and Gram-negative bacteria, respectively. The results show that the epimerase reac- tion follows a nonstereospecific retroaldol ⁄ aldol mech- anism with the same reaction intermediate as that of the aldolase reaction and that SaDHNA and EcDHNA have significantly different equilibrium and kinetic constants, which form the basis for elucidating the catalytic mechanism of DHNA and developing antimicrobial agents specifically against Gram-positive or Gram-negative bacteria. Results NMR analysis Although it is reasonable that the epimerase reaction follows the same reaction intermediate as that of the aldolase reaction, as described above (Fig. 1), it is also possible that it follows an alternative mechanism, i.e. the deprotonation and reprotonation of C2¢. The alter- native reaction can be initiated by deprotonation of C1¢ and protonation of N5 to form an enol intermedi- ate, which can turn into a keto intermediate by tau- tomerization for the subsequent deprotonation and reprotonation of C2¢. Whether the epimerase reaction follows the same reaction intermediate as that of the aldolase reaction or the mechanism of deprotonation and reprotonation of C2¢ can be tested by NMR. The key difference between the two reaction mechanisms is that H2¢ is always attached to C2¢ if the epimerase reaction follows the same reaction intermediate as that of the aldolase reaction (Fig. 1), whereas it has to be extracted by a base if the epimerase reaction follows the mechanism of deprotonation and reprotonation of C2¢. Therefore, when the reaction is run in D 2 O, the H2¢ occupancy will change if the epimerase reaction involves the deprotonation and reprotonation of C2¢, but will not change if it follows the same reaction Fig. 2. Amino-acid sequence alignment of DHNAs. The top five DHNAs are from Gram-positive bacteria: Staphylococcus aureus (SA), Bacillus subtilis (BS), Strepto- coccus pyogenes (SP), Listeria innocua (LI), and Streptomyces coelicolor (SC). The bot- tom six DHNAs are from Gram-negative bacteria: Escherichia coli (EC), Yersinia pes- tis (YP), Vibrio cholerae (VC), Haemophilus influenzae (HI), Pseudomonas aeruginosa (PA), and Shewanella oneidensis (SO). The highly conserved residues among all DHNAs are shaded in black. Residues that are char- acteristic of Gram-positive or Gram-negative bacteria are highlighted in gray. Residues that comprise the active centers are indica- ted by horizontal bars. The residue number- ing at the top of the alignment is that of SaDHNA. Mechanism and kinetics of dihydroneopterin aldolase Y. Wang et al. 2242 FEBS Journal 274 (2007) 2240–2252 ª 2007 The Authors Journal compilation ª 2007 FEBS intermediate as that of the aldolase reaction. The pro- ton occupancy can be quantified by NMR. The result of such an experiment is shown in Fig. 3. The NMR signals were assigned on the basis of their multiplicity patterns, decoupling experiments, and comparison with the NMR spectrum of authentic DHMP (the top spec- trum in Fig. 3). As shown in Fig. 3, the NMR signals of all 2¢ and 3¢ protons of DHNP and DHMP are well separated, except those of the 3¢Hb protons of the two compounds, which are overlapping. The proton occu- pancy at the 2¢ position of the newly formed DHMP could be quantified by comparing the integrals of the 2¢H and 3¢Ha NMR signals of DHMP, because 3¢ pro- tons do not participate in the chemical reaction in either mechanism and cannot be replaced with deuter- ons. The result showed that the intensities of the 2¢H and 3¢Ha NMR signals were the same throughout the time course of the reaction (18, 35, and 70 min). The 1 : 1 intensities of the 2¢H and 3¢Ha NMR signals indi- cated a 100% proton occupancy at the 2¢ position, strongly suggesting that there is no deprotonation and reprotonation at C2¢ and the epimerase reaction fol- lows the aldol chemistry. Is the DHNA-catalyzed reaction reversible? Although aldolase-catalyzed reactions are generally reversible, the DHNA-catalyzed reaction was shown previously to be irreversible [4]. However, it was noticed that the E. coli enzyme preparation used in the experiment had a low activity and furthermore, the glycoaldehyde (GA) concentration (150 lm) was rather low, especially considering that it exists in various forms in solution and only a small fraction is in the correct form for the reaction [19,20]. To further investigate the issue of the reversibility of the DHNA- catalyzed reaction, we ran the reverse reaction with our recombinant enzymes and high concentrations of GA. One such result obtained with SaDHNA is shown in Fig. 4. Clearly, the SaDHNA-catalyzed reaction was reversible. Furthermore, the reverse reaction was rather rapid in the presence of SaDHNA. The appar- ent K m for GA obtained by varying GA at a fixed HP Fig. 3. NMR analysis of the SaDHNA-catalyzed reactions in D 2 O. The bottom spectrum was obtained before the addition of the enzyme, and the middle three spectra were obtained 18, 35, and 70 min after the addition of the enzyme. The top spectrum is that of DHMP for comparison. Only the NMR signals of the 2¢ and 3¢ protons of DHNP and DHMP are shown. The chemical structures of DHNP and DHMP are also shown at the top, with atom number- ing labeled for DHNP. For clarity, the NMR signals of the aldolase reaction products HP and GA are not shown. Fig. 4. HPLC analysis of the reverse reaction catalyzed by SaDHNA. The initial reaction mixture in 100 m M Tris ⁄ HCl, pH 8.3, contained 100 l M HP and 20 mM GA. The reaction was initiated with 10 l M SaDHNA at 25 °C and quenched with 1 M HCl. The reverse reaction generated both DHNP and DHMP. HP, DHNP, and DHMP were oxidized to HPO, NP, and MP, respectively, before the HPLC analysis as described in Experimental procedures. Y. Wang et al. Mechanism and kinetics of dihydroneopterin aldolase FEBS Journal 274 (2007) 2240–2252 ª 2007 The Authors Journal compilation ª 2007 FEBS 2243 concentration (100 lm) was  10 mm. Both DHNP and DHMP were generated in the reverse reaction, which also lent support to a nonstereospecific retroal- dol ⁄ aldol mechanism for the epimerization reaction. Equilibrium binding studies As the epimerase reaction uses the same reaction inter- mediate as that of the aldolase reaction and the aldo- lase reaction is reversible, we can draw a unified kinetic scheme for the DHNA-catalyzed reactions as shown in Scheme 1, where A, B, I, P, and Q represent DHNP, DHMP, the reaction intermediate, HP, and glycolaldehyde, respectively. The major goal of this work was to determine the rate constants of the individual steps of the reactions. Our strategy to achieve this goal was a comprehensive one, involving the measurements of both equilibrium and kinetic constants of the physical steps by equilib- rium and stopped-flow fluorimetric analysis and the determination of the rate constants of the chemical steps by quench-flow analysis of both forward and reverse reactions. We first measured the dissociation constants by fluorimetry. A typical fluorimetric titra- tion curve is shown in Fig. 5. The results are summar- ized in Table 1. To facilitate the purification of SaDHNA, we engineered a His-tag at the N-terminus of the enzyme. The binding properties of the His- tagged and untagged enzymes were essentially the same (data not shown), and the binding data for SaDHNA in Table 1 are those of the His-tagged enzyme. d-Neopterin (NP), l-monapterin (MP), and 6-hydroxy- methylpterin (HPO) are the oxidized forms of DHNP, DHMP, and HP, respectively. The only difference between the two sets of pterin compounds is that the link between C7 and N8 is a single bond in the reduced pterins but a double bond in the oxidized pterins. Consequently, there is a hydrogen atom attached to N8 in the reduced pterins and the NH group can serve as a hydrogen-bond donor, whereas in the oxidized pterins, there is no hydrogen attached to N8 and it can only serve as a hydrogen-bond acceptor. NP, MP, and HPO are all DHNA inhibitors. The binding of the inhibitors to the enzymes cause a decrease in their fluorescence intensities. The increasing fluorescence intensities in Fig. 5A were obtained by subtracting the control titration data in the absence of the enzymes from the titration data in the presence of the enzymes. The results of the equilibrium binding studies showed that, in comparison with EcDHNA, SaDHNA has significantly higher K d values for the measured pterin compounds, particularly HPO, whose the K d value for SaDHNA was 240 times that for E+AEAEIEBE+B EPQ E+P+Q k 6 k -6 k 1 k -1 k 2 k -2 k 5 k -5 k 3 k -4 k 4 k -3 Scheme 1. Kinetic mechanism of the DHNA-catalyzed reactions. A B Fig. 5. Fluorimetric titration of SaDHNA with NP (A) and of HPO with SaDHNA (B). (A) A 2-mL solution containing 15 l M SaDHNA in 100 m M Tris ⁄ HCl, pH 8.3, was titrated with NP by adding aliquots of a 1.94 m M NP stock solution in the same buffer at 24 °C. The final enzyme concentration was 14 l M. The top axis indicates the NP concentrations during the titration. A set of control data was obtained in the absence of the enzyme and was subtracted from the corresponding data set obtained in the presence of the enzyme. (B) A 2-mL solution containing 1 l M HPO in 100 mM Tris ⁄ HCl, pH 8.3, was titrated with SaDHNA by adding aliquots of a 1.55 m M SaDHNA stock solution in the same buffer at 24 °C. The final HPO concentration was 0.93 l M. The top axis indicates the SaDHNA concentrations during the titration. A set of control data was obtained in the absence of HPO and was subtracted from the corresponding data set obtained in the presence of the enzyme. The solid lines were obtained by nonlinear least-squares regression as previously described [25]. Mechanism and kinetics of dihydroneopterin aldolase Y. Wang et al. 2244 FEBS Journal 274 (2007) 2240–2252 ª 2007 The Authors Journal compilation ª 2007 FEBS EcDHNA. Furthermore, whereas the K d values of SaDHNA for the reduced and oxidized pterin com- pounds (HP and HPO, respectively) were the same, the K d value of EcDHNA for the reduced pterin com- pound (the product HP) was higher than that for the oxidized pterin compound (the oxidized product HPO). Finally, the K d value of SaDHNA for NP was slightly higher than that for MP, whereas the K d value of EcDHNA for NP was lower than that for MP. Stopped-flow analysis We then measured the rate constants of the physical steps of the reaction by stopped-flow fluorimetric ana- lysis. Because GA has a very low affinity for the enzymes (data not shown) and exists in solution in multiple forms, of which the correct form for the reac- tion is a minor one [19,20], we focused our analysis of product binding and dissociation on HP. Because DHNP and DHMP undergo chemical reactions in the presence of DHNA, we measured the binding and dis- sociation of the structurally related DHNA inhibitors NP and MP. To assess the differences in the rate con- stants of the reduced and oxidized pterins, we also measured the association and dissociation rate con- stants of HPO and compared them with those of HP. A representative set of the stopped-flow analysis data is shown in Fig. 6. The rate constants measured by the stopped-flow experiments are summarized in Table 2, where k 1 and k )1 are the association and dissociation rate constants, respectively. The K d values calculated as k )1 ⁄ k 1 were in excellent agreement with those meas- ured by equilibrium binding studies (Table 1). The results show that the association rate constants for NP and MP are very similar and slightly lower than those for HP and HPO, which are very similar. This phe- nomenon is presumably related to the sizes of the molecules. NP and MP are the same size and are slightly larger than HP and HPO. Furthermore, the results also show that, for SaDHNA, the association and dissociation rate constants of the reduced pterin HP are the same as those of the oxidized pterin Fig. 6. Stopped-flow analysis of the binding of HPO to SaDHNA. The concentration of SaDHNA was 2 l M, and the concentrations of HPO were 10, 20, 30, and 40 l M for traces 1, 2, 3, and 4, respect- ively. All concentrations were those immediately after the mixing of the two syringe solutions. Both SaDHNA and HPO were dissolved in 100 m M Tris ⁄ HCl, pH 8.3. The fluorescent signals were rescaled so that they could be fitted into the figure with clarity. The solid lines were obtained by nonlinear regression as described in Experi- mental procedures. The inset is a replot of the apparent rate con- stants versus the HPO concentrations. The solid line in the inset was obtained by linear regression. Table 2. Association and dissociation rate constants of S. aureus and E. coli DHNAs measured by stopped-flow experiments. SaDHNA has a His-tag (MHHHHHH) at the N-terminus. The K d val- ues were calculated as k )1 ⁄ k 1 . SaDHNA EcDHNA k 1 (lM )1 Æs )1 ) k )1 (s )1 ) K d (lM) k 1 (lM )1 Æs )1 ) k )1 (s )1 ) K d (lM) NP 0.24 ± 0.01 4.5 ± 0.1 19 0.32 ± 0.02 0.29 ± 0.03 0.88 MP 0.29 ± 0.02 4.2 ± 0.2 15 0.26 ± 0.01 0.58 ± 0.03 2.3 HP 0.47 ± 0.04 13 ± 1 28 0.65 ± 0.08 0.26 ± 0.02 0.4 HPO 0.45 ± 0.02 10 ± 1 24 0.55 ± 0.04 0.062 ± 0.006 0.11 Table 1. Dissociation constants (lM)ofS. aureus and E. coli DHNAs measured by equilibrium binding experiments. SaDHNA a EcDHNA K d(NP) 18 ± 2 0.77 ± 0.06 K d(MP) 13 ± 1 2.6 ± 0.06 K d(HP) 24 ± 0.2 0.43 ± 0.04 K d(HPO) 24 ± 0.2 0.10 ± 0.007 a The chemical structures of the measured compounds are as fol- lows: N N HN N H 2 N O OH OH OH N N HN N H 2 N O OH OH OH MP N N HN N H 2 N O OH NP HPO b SaDHNA has a His-tag at the N-terminus. Y. Wang et al. Mechanism and kinetics of dihydroneopterin aldolase FEBS Journal 274 (2007) 2240–2252 ª 2007 The Authors Journal compilation ª 2007 FEBS 2245 (HPO), in accordance with the same K d value for the two pterin compounds. On the other hand, for EcDHNA, the association rate constants for HP and HPO are essentially the same, but the dissociation con- stant of HP is larger than that of HPO, in agreement with a larger K d value for HP. Finally, the higher K d values are all mainly due to the higher dissociation rate constants. Quench-flow analysis The rate constants of the chemical steps were meas- ured by quench-flow experiments. We ran the forward reaction (the formation of HP) using both DHNP and DHMP as the substrates and the reverse reaction (the formation of DHNP and DHMP) with HP and GA. For the forward reaction, three concentrations each for DHNP and DHMP were used. For the reverse reaction, the concentration of HP was fixed, and eight concentrations of GA were used for the SaDHNA-cat- alyzed reaction and six concentrations of GA for the EcDHNA-catalyzed reaction. Each reaction generated three curves, one each for DHNP, DHMP, and HP. This multitude of quench-flow data was then fitted glo- bally to Scheme 1 by nonlinear least-squares regression using the program dynafit [21]. The enzyme-bound intermediate (EI) was assumed to isomerize to the aldol product HP during the acid quench and therefore treated as HP in the global fitting analysis. The initial values for the physical steps were derived from the stopped-flow analysis described in the previous section. The rate constants for the chemical steps were estima- ted by global fitting with fixed rate constants for the physical steps. Then the dissociation rate constants were allowed to vary by 20% to obtain the best fit of the data via an iterative process. For SaDHNA, both the association and dissociation rate constants of the oxidized pterin HPO (0.45 lm )1 Æs )1 and 10 s )1 , respectively) were virtually the same as those of the reduced pterin HP (0.47 lm )1 Æs )1 and 13 s )1 , respect- ively), suggesting that HPO is an excellent analogue for HP and, by analogy, NP and MP are excellent analogues of DHNP and DHMP for the kinetic study of the physical steps (association and dissociation). Therefore, the rate constants for the binding of DHNP and DHMP were fixed at the values measured for the corresponding oxidized pterins NP and MP during the initial global fitting analysis. For EcDHNA, the associ- ation rate constant of HPO (0.55 lm )1 Æs )1 ) was very similar to that of HP (0.65 lm )1 Æs )1 ), but the dissoci- ation rate constant of HPO (0.062 s )1 ) was about a quarter of that of HP (0.26 s )1 ), suggesting that the oxidation does not have significant effects on the association rate constant but increases the dissociation rate constant by a factor of  4. Therefore, during the initial global fitting of the EcDHNA quench-flow data, the association constants for the binding of DHNP and DHMP were fixed at the values measured for the corresponding oxidized pterins NP and MP, and the dissociation rate constants were fixed at four times the values measured for the corresponding oxidized pterins. With these constraints, the rate constants for the chemical steps were well determined with standard error less than 15% for both SaDHNA-catalyzed and EcDHNA-catalyzed reactions, except the rate con- stants for the interconversion of the enzyme-bound intermediate (Sa.I in Fig. 8) and enzyme-bound prod- ucts (Sa.HP.GA in Fig. 8) in the SaDHNA-catalyzed reaction. The rate constants for the interconversion of Sa.I and Sa.HP.GA are considered to be approximate low limits, because they were sensitive to lower values but not to higher values. This is probably due to their high values relative to those of the rate constants for other steps and the fact that the reaction rate is insen- sitive to this step when its rate constants increase beyond certain high values. Typical results of the for- ward reaction are shown in Fig. 7 for the SaDHNA- catalyzed reaction. The results of the quench-flow analysis are summarized in Fig. 8. For SaDHNA, the epimerase activity is insignificant in comparison with its aldolase activity, the rate-limiting step in the forma- tion of HP is the generation of the reaction intermedi- ate, and the interconversion of Sa.I and Sa.HP.GA is very fast in comparison with other steps. For EcDHNA, in contrast, the epimerase activity is highly significant (comparable to the aldolase activity), the rate-limiting step in the formation of HP is the prod- uct release, and the interconversion of the enzyme- bound intermediate (Ec.I in Fig. 8) and enzyme-bound products (Ec.HP.GA) is much slower than in the SaDHNA-catalyzed reaction. Discussion DHNA catalyzes the cleavage of the bond between C1¢ and C2¢ of DHNP to form HP (an aldolase reaction) and also the formation of DHMP (an epimerase reac- tion) [7]. A nonstereospecific retroaldol ⁄ aldol mechan- ism has been proposed for the epimerization reaction (Fig. 1) [7], but no experimental evidence in support of such a mechanism has been reported, and one cannot exclude a priori an alternative mechanism of deproto- nation and reprotonation of C2¢ for the epimerization reaction. In this work, we considered these two alter- native mechanisms for the epimerization reaction. Our NMR analysis of DHMP generated in the reaction in Mechanism and kinetics of dihydroneopterin aldolase Y. Wang et al. 2246 FEBS Journal 274 (2007) 2240–2252 ª 2007 The Authors Journal compilation ª 2007 FEBS D 2 O clearly indicates that there is no deuteration of C2¢ of the epimerase product. The lack of deuteration of the C2¢ of DHMP is not due to the lack of deute- rons, because it has been shown previously that the 6-hydroxymethyl group of the aldolase reaction prod- uct, HP, can be significantly deuterated (at least half of the –CH 2 – protons of the hydroxymethyl group) if the reaction occurs in D 2 O [16]. Another possibility is that deprotonation and reprotonation occur without the proton exchanging with bulk water. However, de- protonation and reprotonation in the aldolase reaction involve the proton exchanging with bulk water [16]. The residues that function as the general acid and base in the aldolase reaction are probably the same as those in the epimerase reaction. Therefore, it is unlikely that deprotonation and reprotonation in the epimerase reaction occur without the proton exchanging with bulk water. The NMR data strongly support the hypo- thesis that the epimerase reaction follows a nonstereo- specific retroaldol ⁄ aldol mechanism as depicted in Fig. 1 without deprotonation and reprotonation of C2¢. In further support of this mechanism, we demon- strated that both epimers (DHNP and DHMP) can be generated from the aldolase products (HP and GA). We also observed that, in the transient kinetic experi- ments, the epimerization product (DHMP from DHNP or DHNP from DHMP) accumulated more extensively in the early part of the reaction course and decreased in the late part of the reaction course (data not shown). It suggests that the aldolase and epimerase reactions follow the same reaction intermediate. The product distribution is determined by kinetics in the early part of the reaction course and by thermodynam- ics in the late part of the reaction course, and therefore the epimerization product increases early and decreases as the reaction progresses to the equilibrium. Because DHNA catalyzes both aldol and epimeriza- tion reactions and the epimerization product, DHMP, can also be converted into the aldol reaction product, HP, it is particularly important to determine the rate constants for elementary steps if one intends to deter- mine how the enzyme catalyzes both reactions. Fur- thermore, steady-state kinetic analysis is insufficient for DHNA, because the steady-state kinetic parameters cannot adequately describe the two reactions catalyzed by the enzyme and the formation of DHMP will be underestimated because of its conversion into HP. Haussmann and coworkers previously determined the steady-state kinetic constants for EcDHNA [7]. According to the steady-state kinetic data, the epime- rase activity is one-sixth of the aldolase activity, which significantly underestimates the epimerase activity of EcDHNA (see Fig. 8, lower panel). Furthermore, the k cat values for the aldolase and epimerase activities are significantly lower than the rate constants of the chem- ical steps. A critical issue in the kinetic analysis is whether the reaction is reversible or not. Although aldolase-cata- lyzed reactions are in general readily reversible, it has been shown previously that DHNA is an exception and the DHNA-catalyzed reaction is apparently irre- versible [4]. The apparent irreversibility is probably due to the low activity of the enzyme preparation used in the experiment, the low concentration of GA, and the low reaction rate of the EcDHNA-catalyzed reverse reaction. With pure recombinant enzymes and high concentrations of GA, it is clear that the DHNA- catalyzed reaction is reversible. In fact, for SaDHNA, the reverse reaction is much faster than the forward reaction. Fig. 7. Global analysis of the quench-flow data of the SaDHNA-cata- lyzed reaction. Data 1, 2, 3, 7, 8, and 11 were obtained with DHNP as the substrate. Because the commercial DHNP contained a min- ute amount of DHMP, the initial reaction mixtures contained both DHNP and DHMP. The initial DHNP and DHMP concentrations for these data were 29.7 and 0.3, 19.8 and 0.2, 9.9 and 0.1 l M, respectively. Data 4, 5, 6, 9, 10, and 12 were obtained with DHMP as the substrate. The initial DHMP concentrations for these data were 10, 20, and 30 l M, respectively. The enzyme concentration was 20 l M for all reactions. All concentrations were those immedi- ately after the mixing of the two syringe solutions. The buffer con- tained 100 m M Tris ⁄ HCl, pH 8.3, and 5 mM dithiothreitol. Data 1–6 are the concentrations of the aldolase product, HP, and data 7–12 are the concentrations of the epimerase product, MP or NP. The solid lines were obtained by global nonlinear least-squares regres- sion using the program DYNAFIT [21]. For clarity, the changes in the substrate concentrations were not plotted. The data for the reverse reactions, i.e. with HP and GA as the substrates, were not plotted. Y. Wang et al. Mechanism and kinetics of dihydroneopterin aldolase FEBS Journal 274 (2007) 2240–2252 ª 2007 The Authors Journal compilation ª 2007 FEBS 2247 The rate constants of individual steps, as summar- ized in Fig. 8, were determined by a comprehensive strategy using a combination of stopped-flow and quench-flow analyses. The philosophy behind the strat- egy is to isolate the different steps of the reaction whenever possible and design experiments to determine rate constants for the specific steps. We began the comprehensive kinetic analyses by measuring the rate constants of the physical steps (i.e. the binding steps) by stopped-flow fluorimetry. To avoid the chemical reactions, we substituted NP and MP (see Table 1 for their chemical structures) for DHNP and DHMP, respectively, and measured the binding of HP in the absence of GA. NP and MP are the oxidized forms of the pterins, with a double bond between C7 and N8 instead of a single bond as in the reduced pterins (DHNP and DHMP). To assess the differences in the binding rate constants between the closely related pairs of oxidized and reduced pterins, we also measured the rate constants for the binding of HPO, the oxidized form of HP. These measured rate constants are reliable and accurate, because of (a) the high quality of the stopped-flow data as illustrated in Figs 7 and 8 and (b) the consistency between the K d values calculated from the association and dissociation rate constants (Table 2) and those measured by equilibrium titration experiments (Fig. 5 and Table 1). These measured rate constants are also reasonable in that the association rate constants are similar between NP and MP and between HP and HPO in accordance with the similar shapes and sizes between NP and MP and between HP and HPO. The different K d values are proportional to the different values of the dissociation rate constants, as expected. The results also show that for SaDHNA, HP and HPO have essentially the same rate constants, in accordance with the crystal structure of the complex of SaDHNA with HP, which reveals that NH at posi- tion 8 of HP has no hydrogen bond with the protein [8] and suggest that the rate constants for the binding of the corresponding reduced and oxidized pterins to SaDHNA may be essentially the same. For EcDHNA, HP and HPO have very similar association rate con- stants, but their dissociation rate constants are signifi- cantly different. The dissociation rate constant of HP is about four times that of HPO, suggesting that the corresponding reduced and oxidized pterins may have significantly different dissociation rate constants for binding to EcDHNA. The rate constants of the chemical steps were deter- mined by quench-flow experiments. Because the reac- tion is reversible, we were able to run the reaction in all three directions with DHNP, DHMP, or HP and GA as the substrate(s) so that both forward and reverse rate constants could be defined. Because the three pterin components of the reaction mixtures could be resolved by HPLC (Fig. 4), each set of the quench- flow experiments generated three sets of data. The rate constants of the chemical steps were evaluated by the Fig. 8. Summary of the kinetic constants for the SaDHNA-catalyzed (top panel) and EcDHNA-catalyzed (lower panel) reactions. Sa and Ec represent SaDHNA and EcDHNA, respectively. I represents the reaction inter- mediate as shown in Fig. 1. The rate con- stants for the interconversion of Sa.I and Sa.HP.GA are considered to be approximate low limits, and the standard errors for other rate constants are within 15%. Mechanism and kinetics of dihydroneopterin aldolase Y. Wang et al. 2248 FEBS Journal 274 (2007) 2240–2252 ª 2007 The Authors Journal compilation ª 2007 FEBS global fitting of the multitude of the quench-flow data (total nine sets) using the widely used program for kin- etic analysis dynafit [21], which uses the numerical integration of simultaneous first-order ordinary differ- ential equations to calculate the time-course of the chemical reaction and the Levenberg–Marquardt algo- rithm for nonlinear regression fitting. Such transient kinetic analysis has been the standard method for the determination of individual rate constants of enzymatic reactions [22–24] and has been successfully used in our transient kinetic analysis of E. coli 6-hydroxymethyl- 7,8-dihydropterin pyrophosphokinase [25] and yeast cytosine deaminase [26]. Under the reaction conditions, the quench flow data are sensitive to both the physical and chemical steps of the enzymatic reaction and are insufficient for the deter- mination of the rate constants for both the physical and chemical steps. However, when the rate constants of the physical steps are available, the quench-flow data can be used to determine the rate constants of the chemical steps. The rate constants for the physical steps can be estimated from the stopped-flow measurements of the pterin analogues. As the rate constants for the binding of the pair of the reduced and oxidized pterins to SaDHNA are essentially the same, the rate constants for the physical steps of the SaDHNA-catalyzed reac- tion (the first step in each direction) are well defined. For the EcDHNA-catalyzed reaction, the association rate constants for the physical steps were assumed to be the same as those for the binding of the oxidized pterins (NP and MP), because the oxidation has no significant effects on the association rate constants, and the stereo- chemistry of the trihydroxypropyl tail has no significant effects either. The dissociation rate constants for DHNP and DHMP were estimated from those for NP and MP and the difference between HP and HPO and finalized by iterative fittings as described in the Results section. When the rate constants for the physical steps were fixed, the rate constants for the chemical steps were well defined in the sense that > 15% variations in the rate constants, except those for the conversion of the reaction intermediate into the aldolase products (HP and GA) in the SaDHNA-catalyzed reaction, would have significant detrimental effects on the fittings. The rate constants for the conversion of the reaction intermediate into the aldolase products in the SaDHNA-catalyzed reaction must be considered to be the low limits, because decreasing the values of these rate constants had significant detrimental effects but increasing the values of these rate constants had insigni- ficant effects on the fittings. Our equilibrium and kinetic data also show that SaDHNA and EcDHNA have significantly different binding and catalytic properties, in accordance with the significant sequence differences between the two enzymes. EcDHNA is biochemically different from SaDHNA in several aspects. (a) EcDHNA has much higher affinities for the substrate, products, and inhibi- tors as measured in this work, particularly for HPO. (b) EcDHNA has a much higher epimerase activity than SaDHNA. (c) The rate-limiting step in the for- ward reaction (the formation of HP) is the product release for EcDHNA but is the formation of the reaction intermediate for SaDHNA. (d) The intercon- version of the enzyme-bound intermediate and enzyme-bound aldolase products is much slower in the EcDHNA-catalyzed reaction than in the SaDHNA- catalyzed reaction. The marked differences in the lig- and-binding properties of SaDHNA and EcDHNA, which must stem from the significant differences in the structures of their active sites, suggest that it may be possible to develop antimicrobial agents specifically against DHNA from S. aureus or E. coli. Because many DHNAs from Gram-positive and Gram-negative bacteria are highly homologous within their own groups but significantly different between the two groups, it may be possible to develop antimicrobial agents specifically against Gram-positive or Gram- negative bacteria by targeting respective DHNAs. Experimental procedures Materials HPO, HP, DHNP, DHMP, NP, and MP were purchased from Schircks Laboratories (Jona, Switzerland). Restriction enzymes and T4 ligase were purchased from New England Biolabs (Ipswich, MA, USA). Pfu DNA polymerase and the pET-17b vector were purchased from Stratagene (La Jolla, CA, USA) and Novagen (Madison, WI, USA), respectively. Other chemicals were from Sigma-Aldrich (St Louis, MO, USA). Cloning The SaDHNA gene was cloned into the prokaryotic expression vector pET-17b and a home-made derivative (pET17H) by PCR from S. aureus genomic DNA. The pET17H vector was used for the production of a His- tagged SaDHNA. The primers for the PCR were 5¢-GG AATTCCATATG CAAGACA CAAT CTTTCTT AAAG-3¢ (forward primer with a Nde I site) and 5¢-CGGGATCCT CATTTATTCTCCCTCACTATTTC-3¢ (reverse primer with a BamHI site). The EcDHNA gene was cloned into the prokaryotic expression vector pET-17b by PCR from E. coli genomic DNA. The primers for the PCR were Y. Wang et al. Mechanism and kinetics of dihydroneopterin aldolase FEBS Journal 274 (2007) 2240–2252 ª 2007 The Authors Journal compilation ª 2007 FEBS 2249 [...]... wavelength of 446 nm and an excitation wavelength of 360 nm for HPO The emission and excitation slits were both 5 nm A control titration experiment was performed in the absence of the ligand The control data set obtained in the absence of the ligand was subtracted from the corresponding data set obtained in the presence of the ligand The Kd values were obtained by nonlinear least-squares fitting of the titration... wavelength of 446 nm with a slit of 5 nm using a Spex FluoroMax-2 fluorimeter The excitation wavelength and slit were 400 nm and 1 nm, respectively A set of control data was obtained in the absence of the protein The data set obtained in the absence of the protein was then subtracted from the corresponding data set obtained in the presence of the protein after correction of inner-filter effects The Kd value... fitting of the titration data as previously described [25] The Kd values for HP and HPO were determined by titrating a ligand solution with the proteins Aliquots of a protein stock solution were added to a ligand solution The fluorescence of the ligand was measured after each addition of the protein stock solution at an emission wavelength of 430 nm and an excitation wavelength of 330 nm for HP and at... with D2O The reaction was initiated with 3 lm SaDHNA NMR spectra were recorded before and after the addition of the enzyme A spectrum of DHMP was also acquired Mechanism and kinetics of dihydroneopterin aldolase for comparison The spectral width for the NMR data was 8000 Hz with the carrier frequency at the HDO resonance The solvent resonance was suppressed by presaturation Each FID was composed of 16k... proteins and ligands were all dissolved in 100 mm Tris ⁄ HCl, pH 8.3, and the titration experiments were performed in a single cuvette at 24 °C The Kd values for NP and MP were determined by titrating a protein solution with the ligands Aliquots of a stock solution of one of the ligands was added to the protein solution Fluorescence intensities were measured after each addition of the ligand stock... Biosynthesis of pteridines in Escherichia coli: structural and mechanistic similarity of dihydroneopterin- triphosphate epimerase and dihydroneopterin aldolase J Biol Chem 273, 17418–17424 8 Hennig M, D’Arcy A, Hampele IC, Page MGP, Oefner C & Dale GE (1998) Crystal structure and reaction FEBS Journal 274 (2007) 2240–2252 ª 2007 The Authors Journal compilation ª 2007 FEBS 2251 Mechanism and kinetics of dihydroneopterin. .. spectrofluorimeter (Leatherhead Surrey, UK) at 25 °C One syringe contained the protein (SaDHNA or EcDHNA), and the other contained NP, MP, HP or HPO The protein concentrations were 1 or 2 lm, and the ligand concentrations ranged over 5–60 lm All concentrations were those after the mixing of the two syringe solutions Fluorescence traces for NP, MP and HPO were obtained with an excitation wavelength of 360 nm and a filter... structure of 7,8-dihydrobiopterin bound to dihydroneopterin aldolase J Biol Chem 275, 30139–30143 Illarionova V, Eisenreich W, Fischer M, Haussmann C, Romisch W, Richter G & Bacher A (2002) Biosynthesis of tetrahydrofolate: stereochemistry of dihydroneopterin aldolase J Biol Chem 277, 28841–28847 Yao LS, Yan HG & Cukier RI (2006) Mechanism of dihydroneopterin aldolase: a molecular dynamics study of the apo.. .Mechanism and kinetics of dihydroneopterin aldolase Y Wang et al 5¢-GGAATTCCATATGGATATTGTATTTATAGAGCA AC-3¢ (forward primer with a Nde I site) and 5¢-CGGGA TCCTTAATTATTTTCTTTCAGATTATTGCC-3¢ (reverse primer with a BamHI site) The expression constructs were transformed into the E coli strain DH5a The correct coding sequences of the cloned genes were verified by DNA sequencing The verified SaDHNA... 16 transients The delay between successive transients was 6 s The time domain data were processed by zero-filling to 32k points, multiplication with a 90°-shifted sine bell function, and Fourier transformation Chemical shifts were referenced to the internal standard sodium 2-dimethyl-2silapentane-5-sulfonate sodium salt The relative proton populations were calculated on the basis of the integrals of their . Mechanism of dihydroneopterin aldolase NMR, equilibrium and transient kinetic studies of the Staphylococcus aureus and Escherichia coli enzymes Yi. because of the lack of three enzymes in the middle of the folate pathway, and they therefore obtain folates from the diet. DHNA is the first of the three enzymes