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Narrow substrate specificities often limit the use of enzymes in biocatalysis. To further the development of Escherichia coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase as a biocatalyst, the molecular determinants of substrate specificity were probed by mutagenesis. Our data demonstrate that S184 is located in the substrate-binding pocket and interacts with the phosphate moiety of KDPG, providing biochemical support for the binding model proposed on the basis of crystallographic data. An analysis of the substrate selectivity of the mutant enzymes indicates that alterations to the phosphate-binding site of KDPG aldolase changes the substrate selectivity. We report mutations that enhance catalysis of aldol cleavage of substrates lacking a phosphate moiety and demonstrate that electrophile reactivity correlates with the hydrophobicity of the substituted side chain. These mutations improve the selectivity for unnatural substrates as compared to KDPG by up to 2000-fold. Furthermore, the S184L KDPG aldolase mutant improves the catalytic efficiency for the synthesis of a precursor for nikkomycin by 40-fold, making it a useful biocatalyst for the preparation of fine chemicals.
The development of catalysts that effect synthetic transformations with predictable behavior is among the most challenging task facing synthetic chemistry. Biocatalysis—the use of enzymes to carry out synthetic transformation—is a field dating to the late nineteenth century. Enzymes are especially attractive catalysts for organic synthesis, given their ability to catalyze reactions with remarkable efficiency, substrate specificity, and stereoselectivity. The broader use of enzymes in biocatalysis is limited by the defined substrate specificities displayed by enzymes derived from natural sources; the de novo design of protein catalysts with predictable, defined substrate specificities is a vigorous area of research. Despite over 30 years of effort in the field, an ability to rationally redesign enzyme active sites remains only rudimentary, a reflection of the complex structural basis of enzyme activity.
The aldolases are a large group of enzymes that catalyze reversible stereoselective aldol addition and have demonstrated utility in organic synthesis (Fessner and Walter 1997; Samland and Sprenger 2006). 2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase is an especially attractive enzyme for development as a biocatalyst, showing good thermal stability (Griffiths et al. 2002), broad pH optimum, high tolerance of organic cosolvent (Shelton et al. 1996), high stereoselectivity, moderately broad substrate specificity, and ready availability. Together, these properties make KDPG aldolase a prime candidate for use as a catalyst in a number of synthetic applications (Henderson et al. 1997; Cotterill et al. 1998).
In vivo, KDPG aldolase functions as part of the Entner–Doudoroff glycolytic pathway, catalyzing the reversible retro-aldol cleavage of KDPG to pyruvate and D-glyceraldehyde-3-phosphate (GAP) (Fig. 1; Peekhaus and Conway 1998). The reverse reaction, the stereospecific formation of a carbon–carbon bond between pyruvate and a range of electrophilic aldehydes, is of significant interest for the synthesis of fine chemicals. However, as with most enzymatic catalysts, substrate selectivity ultimately limits the broad use of KDPG aldolase as a synthetic reagent. Recently, both rational design and directed evolution have been used to alter the substrate specificity of various aldolases with improvements in kcat/KM of up to 25-fold (DeSantis et al. 2003; Wada et al. 2003; Franke et al. 2004; Hsu et al. 2005; Ran and Frost 2007); similar efforts to alter the substrate specificity of KDPG aldolase have also been reported (Fong et al. 2000; Wymer et al. 2001). Further rational redesign of KDPG aldolase is limited by the lack of a detailed model of the substrate-binding site. We have reported crystallographic studies of various bound forms of KDPG aldolase from Thermatoga maritima (Fullerton et al. 2006) and Escherichia coli (Wymer et al. 2001), both of which crystallized bound to a dibasic inorganic anion. With the assumption that this ion reveals the phosphate-binding site, we proposed a binding model for full length substrate (Fig. 2). Here, we provide biochemical evidence for the validity of this model and consider the identity of the binding site for the electrophilic substrate through the preparation of a series of mutant proteins. We demonstrate that alteration of the phosphate-binding pocket significantly changes the substrate selectivity of this enzyme, providing biocatalysts with increased activity toward nonphosphorylated electrophiles. Furthermore, the mutant enzymes described here are useful for the synthesis of nikkomycins (Henderson et al. 1997), a group of potent inhibitors of chitin synthase (Schlüter 1982). These studies provide an essential background for structure-based remodeling of the KDPG aldolase active site and for the rational development of novel biocatalysts with predetermined substrate specificities.
Figure Figure 2.. Model of KDPG bound to KDPG aldolase. Structural model of KDPG bound to KDPG aldolase based on the crystal structure of T. maritima aldolase (IWA3; blue) and the E. coli aldolase (1EUA; red). KDPG bound as a Schiff base was modeled as described by Fullerton et al. (2006). Residues that line the phosphate-binding site G162, G163, I164, and S184 are shown with possible hydrogen bond contacts to the substrate drawn with yellow lines. The catalytic residues E45 and K133 are also shown.
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The first step in both aldol addition and retro-aldol cleavage by KDPG aldolase is the formation of a Schiff-base between an active site lysine and the C2 carbonyl of the nucleophilic substrate (Fig. 3). In E. coli KDPG aldolase this residue is K133, an assertion verified both by crystallographic analysis of pyruvate-bound protein (both Schiff base and carbinolamine intermediates) (Allard et al. 2001; Fullerton et al. 2006) and by mutagenesis (Wymer et al. 2001). A general base catalyzes the formation of the nucleophilic enamine, which, in turn, reacts with the aldehydic carbon of the electrophilic substrate, glyceraldehyde-3-phosphate, to form KDPG. Glutamate 45, located proximal to Lys133 in space, was proposed as the catalytic general acid/general base, functioning through the intermediacy of bound water molecules (Fullerton et al. 2006).
The structure of KDPG aldolase, both apoenzyme and various bound forms, has been studied extensively (Mavridis et al. 1982; Allard et al. 2001; Wymer et al. 2001; Fullerton et al. 2006). Despite a wealth of crystallographic data regarding the location of the pyruvate-binding site, little structural or biochemical information exists that informs the location or nature of the glyceraldehyde-3-phosphate-binding site. A plausible location of the electrophilic binding site was proposed based on crystal structures of the E. coli and T. maritima KDPG aldolases, both of which crystallize with sulfate ions bound to a solvent-exposed cleft roughly 10 Å from the catalytic residues (Fig. 2; Allard et al. 2001; Fullerton et al. 2006). The site is a highly conserved region of KDPG aldolase and is formed by G162, G163, I164, and S184. These residues are found in the loop regions between β-sheet 7/helix 7 and β-sheet 8/helix 8, a location where phosphate binding motifs are observed in other TIM barrel proteins (Wilmanns et al. 1991; Nagano et al. 2002). Based on these data, we proposed a model of full-length substrate binding that locates the KDPG phosphate moiety in the place of the sulfate ions observed in pyruvate-bound aldolase. To test the validity of this model we examined the effect of perturbing one of the residues in this cleft, serine 184, on the reactivity of this enzyme.
The sulfate ion observed in the crystal structure forms hydrogen bonds with the backbone amides of G162, G163, I164, and the side chain hydroxyl of S184. The two glycine residues create a tight turn between β-sheet 7 and helix 7; mutation of these residues would likely cause significant structural reorganization, precluding these sites as targets for mutagenesis. We thus considered the functional effect of altering S184, changing the residue to alanine, leucine, and aspartate. Alanine mutation probes the role of hydrogen bonding, while the leucine substitution explores the effect of partially occluding the binding site with a hydrophobic residue. Finally, mutation of position 184 to aspartate investigates the effect of inserting negative charge into the putative phosphate-binding pocket. The functional consequences of these mutations are quantified by evaluating the effect of the mutation on catalysis of aldol cleavage of several substrates.
The steady-state kinetic parameters for KDPG cleavage catalyzed by each mutant enzyme were determined to analyze the importance of the interaction of the phosphate moiety with the phosphate-binding pocket. Kinetic parameters were also determined for the cleavage of 2-keto-3-deoxy-gluconate (KDG), an analog of KDPG that lacks the C6 phosphate, to isolate effects that are specific to an interaction with the phosphate moiety. And for 2-keto-4-hydroxyoctonate (KHO), a substrate analog in which the C5 alcohol and C6 phosphate are replaced with a hydrogen and an ethyl group, respectively, was studied to probe the potential for new hydrophobic interactions between substrates and the side chain at position 184 that could enhance catalytic activity toward unnatural substrates (Fig. 4; Table 1).
Table Table 1.. Kinetic parameters catalyzed by S184 mutants of E. coli KDPG aldolase
Figure Figure 4.. Substrates for KDPG aldolase. (A) KDPG is the natural substrate. (B) 2-keto-3-deoxygluconate (KDG) lacks the C6 phosphate. (C) 2-keto-4-hydroxy-octonate (KHO) is a hydrophobic analog of KDPG. (D) 2-keto-4-hydroxy-4-(2′-pyridyl)butyrate (KHPB) is a reactive analog. (E) 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) differs from KDPG by only the stereochemistry at C4.
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Wild-type KDPG aldolase efficiently catalyzes retro-aldol cleavage of KDPG with values of kcat/KMKDPG and kcat of 8.3 × 105 M−l s−1 and 83 s−1, respectively. Removal of the C6 phosphate in the substrate (KDG) decreases the catalytic efficiency of KDPG aldolase enormously, by 8.6 × 104-fold, a loss due to both a 520-fold decrease in the value of kcat and a 160-fold increase in the value of KM. The efficiency of retro-aldol cleavage of the hydrophobic substrate analog KHO is comparable to that of KDG, with a decrease in kcat/KM of 6 × 104 relative to KDPG. In this instance, however, the loss in activity is primarily the result of a 1400-fold increase in KM, presumably reflecting mainly weaker substrate binding.
Mutation of S184 in KDPG aldolase to alanine decreases the efficiency of KDPG retro-aldol cleavage only modestly, reducing kcat/KMKDPG by 27% to 6.1 × 105 M−l s−1. Deletion of this single interaction with the phosphate group clearly does not recapitulate the low catalytic activity of the KDG substrate, presumably reflecting the ability of the remaining residues in the putative phosphate-binding site to effectively bind and orient the substrate for catalysis. The S184A mutation has only a modest effect on the catalytic efficiency for cleavage of KDG, and increases the value of kcat/KMKHO for cleavage of KHO by threefold.
Similar trends in reactivity are observed for the S184L mutation, although the magnitude of the effect is larger; the catalytic efficiency of KDPG cleavage decreases nearly fourfold as compared to the wild-type enzyme. This effect is due mainly to a threefold increase in KM, suggesting that the leucine substitution further disrupts interactions with the phosphate-binding pocket. In contrast to the S184A mutant, S184L KDPG aldolase increases the efficiency for retro-aldol cleavage of both KDG and KHO, with improvements in kcat/KM, of 5.5- and 8-fold, respectively. The increase in efficiency for KDG cleavage occurs mainly through an increase in kcat while for KHO cleavage the KM is reduced. Therefore, the S184L mutant has an enhanced selectivity for KHO over KDPG of 32-fold relative to wild-type KDPG aldolase11 (Table 2).
Table Table 2.. Improvements in selectivity for unnatural substrates as compared to KDPG
In contrast to the modest effects of the Ala and Leu substitutions, mutation of S184 to aspartate significantly abrogates KDPG cleavage activity, diminishing kcat/KMKDPG by over 300-fold; both KM and kcat are affected, by 12- and 26-fold, respectively. Surprisingly, this mutation increases the catalytic efficiency of aldol cleavage of substrates lacking a negatively charged phosphate group; the values of kcat/KM for catalysis of KDG and KHO cleavage increase by 6.5- and 2.5-fold, respectively, as compared to the wild-type enzyme. The S184D mutant also enhances the selectivity for reaction with KHO compared to KDPG by 780-fold relative to the wild-type KDPG aldolase (Table 2).
To further consider the effect of mutations to the phosphate-binding pocket on the reactivity and selectivity of KDPG aldolase, we measured steady-state kinetic parameters for retro-aldol cleavage of 2-keto-4-hydroxy-4-(2′-pyridyl)butyrate (KHPB). KDPG aldolase efficiently cleaves this substrate (Henderson et al. 1997) despite the structural dissimilarity to KDPG (Fig. 4). Kinetic analysis of cleavage of KHPB catalyzed by wild-type KDPG aldolase (Table 3) indicates that kcat/KMKHPB is reduced by 2500-fold compared to that of KDPG; however, the catalytic efficiency for this substrate is still more than one order of magnitude greater than either KDG or KHO. Aldol addition proceeds with si-facial stereochemical specificity, identical to natural substrates (Henderson et al. 1998), suggesting that KHPB and KDPG may share a common binding orientation. The rapid turnover of this substrate presumably reflects, at least in part, the intrinsic reactivity of the electrophilic pyridyl-aldehyde.
Table Table 3.. Kinetic parameters for cleavage of KHPB catalyzed by E. coli KDPG aldolase mutants
Mutation of S184 to A, L, or D enhances the catalytic efficiency for retro-aldol cleavage of KHPB by 6- to 40-fold and with the same order of reactivity observed for catalysis of KHO cleavage, i.e., S184L > S184A > S184D > wild type. All of the mutations improve KM by 10-fold relative to wild-type enzyme. Remarkably, the S184L mutation also enhances turnover fourfold, which, coupled with the tighter apparent binding affinity, leads to a 40-fold enhancement in catalytic activity against this substrate. The S184D mutation also significantly alters the substrate selectivity such that the values of kcat/KM are comparable for KHPB and KDPG (Tables 1 and 3).
The turnover number of the S184L enzyme for cleavage of KHPB is sufficiently rapid to make this enzyme a viable candidate for practical biocatalyst development. To further examine the suitability of S184L KDPG aldolase as a biocatalyst, we evaluated the stereoselectivity of enzyme-catalyzed aldol addition of pyruvate to 2-pyridine carboxaldehyde. The KHPB product was converted to the corresponding dithiolactone and analyzed by chiral GC, which showed a single peak. These data demonstrate that the S184L mutant behaves like wild type (Henderson et al. 1998) with regard to enantioselectivity, achieving greater than 99.7% si-facial addition product. The ability of this mutant to more efficiently catalyze KHPB cleavage and yet still retain precise stereochemical control makes it an excellent candidate as a practical biocatalyst for the synthesis of nikkomycins.
To further analyze the effect of mutations at S184 on the stereoselectivity of KDPG aldolase, we measured the steady-state kinetic parameters for retro-aldol cleavage of 2-keto-3-deoxy-6-phosphogalactonate (KDPGal; Table 4). Since KDPGal and KDPG differ only by the identity of the chiral center at C4 (Fig. 4), the enantioselectivity of each enzyme can be measured by the ratio of (kcat/KMKDPG)/(kcat/KMKDPGal). Wild-type KDPG aldolase has an enantiomeric selectivity for cleavage of KDPG compared to KDPGal of 1.4 × 104-fold. The S184L mutation in KDPG aldolase causes a larger decrease in the value of kcat/KMKDPGal than the value of kcat/KMKDPG, thereby increasing the enantiomeric selectivity ratio to 7.8 × 104 (Table 4). The S184D mutant has an enantiomeric selectivity ratio of 1.1 × 104, indicating that the stereoselectivity of this mutant is the same as wild type within error. These data confirm that mutation at S184 does not directly alter the stereoselectivity of KDPG aldolase.
Table Table 4.. Kinetic parameters for cleavage of KDPGal catalyzed by E. coli KDPG aldolase mutants and the enantiomeric selectivity ratio