A novel genetic selection system for PLP-dependent threonine aldolases

Tetrahedron 68 (2012) 7549e7557

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A novel genetic selection system for PLP-dependent threonine aldolases
re d, Donald Hilvert a, * Lars Giger a, y, Miguel D. Toscano a, b, y, Madeleine Bouzon c, Philippe Marlie € rich, 8093 Zurich, Switzerland Laboratory of Organic Chemistry, ETH Zu Novozymes A/S, Krogshøjvej 36, 2880 Bagsvaerd, Denmark c CEA, DSV, IG, Genoscope, 2 rue Gaston Cr emieux, CP5706, F-91057 Evry Cedex, France d Heurisko USA Inc., 113 Barksdale Professional Center, Newark, DE 197113258, USA a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 April 2012 Accepted 22 May 2012 Available online 28 May 2012

Threonine aldolases are versatile pyridoxal-50 -phosphate (PLP)-dependent enzymes key to glycine, serine and threonine metabolism. Because they catalyze the reversible addition of glycine to an aldehyde to give b-hydroxy-a-amino acids, they are also attractive as biotechnological catalysts for the diastereoselective synthesis of many pharmaceutically useful compounds. To study and evolve such enzymes, we have developed a simple selection system based on the simultaneous inactivation of four genes involved in glycine biosynthesis in Escherichia coli. Glycine prototrophy in the deletion strain is restored by expression of a gene encoding an aldolase that converts b-hydroxy-a-amino acids, provided in the medium, to glycine and the corresponding aldehyde. Combinatorial mutagenesis and selection experiments with a previously uncharacterized L-threonine aldolase from Caulobacter crescentus CB15 (Cc-LTA) illustrate the power of this system. The codons for four active site residues, His91, Asp95, Glu96, and Asp176, were simultaneously randomized and active variants selected. The results show that only His91, which p-stacks against the PLP cofactor and probably serves as the catalytic base in the carbon-carbon bond cleavage step, is absolutely required for aldolase activity. In contrast, Asp176, one of the most conserved residues in this enzyme superfamily, can be replaced conservatively by glutamate, albeit with a >5000-fold decrease in efficiency. Though neither Asp95 nor Glu96 is catalytically essential, they appear to modulate substrate binding and His91 activity, respectively. The broad dynamic range of this novel selection system should make it useful for mechanistic investigations and directed evolution of many natural and artificial aldolases. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Genetic complementation Pyridoxal-50 -phosphate Aldolase Combinatorial mutagenesis Catalysis Mechanism Directed evolution

1. Introduction Pyridoxal-50 -phosphate (PLP) is a strikingly versatile biological cofactor. PLP-dependent enzymes catalyze a wide range of epimerizations, decarboxylations, transaminations, eliminations, and aldol condensations, most notably in basic amino acid metabolism.1,2 In all cases, the cofactor covalently binds the substrate via a Schiff base and stabilizes carbanionic reaction intermediates generated during catalysis. The protein scaffold enhances the intrinsic reactivity of PLP and also determines both substrate preference and reaction type. Though generally assignable to one of five structurally distinct protein families,3,4 individual PLP enzymes are highly divergent with respect to primary sequence and function. For example, the largest family, the Fold Type I or aspartate aminotransferase (AAT) enzymes, encompasses many different reaction types, including the reversible aldol cleavage of b-hydroxy-a-amino acids by threonine aldolases to give glycine and an aldehyde. In living cells, threonine * Corresponding author. E-mail address: [email protected] (D. Hilvert). y These authors contributed equally to this work. 0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tet.2012.05.097

aldolase enzymes are involved in glycine, serine, and threonine metabolism. In the laboratory, they have been exploited biotechnologically for the diastereoselective synthesis of b-phenylserine analogs,5e8 valuable as building blocks for therapeutic agents and other pharmaceutical products. Efforts to expand the substrate scope and enhance the selectivity of PLP-dependent aldolases would benefit from a better understanding of their mechanism of action. Combinatorial mutagenesis, coupled with a suitable screen or selection assay, is a particularly powerful strategy for dissecting protein structure and function.9,10 Although several screening methods have been developed for the directed evolution of improved threonine aldolases,7 their throughput is generally low. Alternatively, given the importance of threonine aldolases to core metabolism, it should be possible to link retro-aldolase activity directly to cellular growth via production of an essential metabolite, such as glycine. Such a system would be greatly advantageous over conventional screens in enabling comprehensive analysis of far larger protein libraries. This article describes a tunable bacterial selection system for threonine aldolases based on an engineered glycine auxotroph. Its

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utility is demonstrated with selection experiments that probe the roles of conserved active site residues in a previously uncharacterized low specificity L-threonine aldolase from Caulobacter crescentus CB15 (Cc-LTA). The sensitivity of growth rate to the structure of the b-hydroxy-a-amino acids used as glycine precursors additionally provides direct insight into the substrate preferences of the target enzyme. Together, our results demonstrate the significant potential of this selection system as a tool for investigating enzyme mechanism and designing new aldolases with tailored properties. 2. Results 2.1. Database search Due to the rapid pace of genomic sequencing, a wealth of data is now available for Fold Type I PLP-dependent enzymes.1,2 Despite divergent function and low sequence identity, members of this protein superfamily exhibit high structural homology. In addition to a Schiff base-forming lysine, which is a common component of all PLP-dependent enzymes, they share a conserved aspartic acid residue that protonates the pyridine ring of the cofactor, making it a better electron sink.11 Hydrophobic residues that pack against the PLP ring and polar residues that interact with the phenolic oxygen and phosphate group of the cofactor and the carboxylate moiety of the substrate are also conserved but less so than the signature lysine and aspartate, reflecting the divergent functions of these catalysts.12 To gain detailed insight into the features that distinguish the aldolase subclass of Fold Type I enzymes, we prepared a multiple sequence alignment of available L-threonine aldolases. At the time of our search, the UniProt database13 of the European Bioinformatics Institute (EBI) included 1408 sequences of putative and assigned PLPdependent aldolases subdivided into clusters with members possessing up to 90% sequence identity.14 The UniRef50 database (http:// www.ebi.ac.uk/uniref/) was used to limit the pairwise sequence identity within each cluster to less than 50%, reducing the total number of non-redundant entries to 216. Subsequent removal of close homologs from different clusters, as well as predicted D-threonine aldolases, alanine racemases and tryptophan synthases, afforded 95 unique PLP-dependent aldolases from all three kingdoms of life. As summarized in Table 1, only five residues are >98% conserved across the entire L-threonine aldolase family. They constitute a subset of the 15 invariant residues identified in an earlier sequence analysis of 16 L-threonine aldolases and 2 alanine racemases,15 and presumably represent the catalytic core of this enzyme family. In addition to the critical lysine and aspartate residues, these include the glycine immediately downstream of the aspartate, a histidine that p-stacks against the re face of the cofactor, and an arginine that forms a salt bridge with the substrate carboxylate in the external aldimine intermediate. These residues are highlighted in Fig. 1, where the active sites of L-threonine aldolases from Thermotoga maritima (Tm-LTA) and Leishmania major (Lm-TA) and a b-phenylserine aldolase from Pseudomonas putida (Ps-LPA) are superimposed. In the Tm-LTA structure complexed to the external aldimine with L-allo-threonine,16 the conserved histidine is within hydrogen bonding distance of the substrate alcohol Table 1 Conserved residues in the L-threonine aldolase familya Cc-LTA residue numberb 66 91 96 176 177 178 179 199 207 290 Consensus residue G H E D G A R D K N 92 100 95 100 100 92 100 96 98 93 % Conservationc a Only residues that were >90% conserved in the 95 analyzed L-threonine aldolase sequences are shown; see Supplementary data for complete alignment. b Amino acid residues targeted for detailed investigations are shown in bold. c The % conservation values (bottom line) are shown in italics to distinguish them from the numbers designating sequence positions (top line).

Fig. 1. The active site of PLP-dependent aldolases. Superposition of conserved residues in the structurally characterized low specificity threonine aldolases from L. major (PDB: 1SVV) and T. maritima (PDB: 1LW4) and a b-phenylserine aldolase from P. putida (PDB: 1V72). The PLP cofactor, bound either as an internal aldimine with the active site lysine or as an external aldimine with L-allo-threonine, is shown in gray sticks. An aspartate (green) forms a salt bridge with the pyridinium nitrogen of the cofactor. The Schiff base-forming lysine (cyan) sits below and a histidine (yellow) sits above the PLP plane. The latter is within hydrogen bonding distance of the substrate hydroxyl group and could serve as the catalytic base; it is oriented via a hydrogen bond with a glutamate (salmon). An arginine (purple) is utilized to bind the substrate carboxylate via a salt bridge.

group, making it a likely candidate for the catalytic base that initiates carbon-carbon bond cleavage. Another five residues are found in 90-96% of the sequences. With the exception of a surface aspartate (Asp199), they either interact directly with the cofactor (Gly66 and Ala178) or with other active site residues (Glu96 and Asn290). Residue Glu96 is of particular note in this context because it hydrogen bonds to His91, favoring the imidazole tautomer that would be necessary for deprotonation of the b-hydroxyl group in the external aldimine intermediate. Although the high conservation of the His-Glu dyad was also evident in previously published alignments and crystal structures of threonine aldolases,15,17 an active functional role in catalysis other than cofactor binding has not been demonstrated. The interaction of this dyad with the side chain of the threonine substrate is reminiscent of the well-known Ser-His-Asp catalytic triad in serine proteases,18 and could conceivably be a catalytically important reaction archetype for aldolase activity. From the complete set of aldolase sequences we chose an uncharacterized, low specificity L-threonine aldolase from C. crescentus CB15 (Cc-LTA) for closer scrutiny. It possesses all 10 consensus residues in the threonine aldolase cluster (Table 1) and is an ideal test case for examining the roles of conserved active site features. 2.2. Construction of the homology model of Cc-LTA Because the structure of Cc-LTA has not been determined, we used the SWISS-MODEL workspace (http://swissmodel.expasy.org/ workspace/) to generate a homology model. The P. putida enzyme Ps-LPA (PDB 1V72), which exhibits 34% sequence identity and is the closest structurally characterized homolog, served as the template structure. Because the fully automated homology model server does not currently support ligands, the PLP cofactor and metal ions were removed from the Pp-LPA PDB file prior to submission of the Cc-LTA sequence. Once the automatically generated model of the apo Cc-LTA enzyme was available, the cofactor bound as a Schiff base to L-allothreonine was docked into the active site by hand using the position of its counterpart in the Pp-PSA template as a guide. Like Ps-LPA, Cc-LTA is a tetramer. Although low sequence identity limits the accuracy with which the tetrameric assembly

L. Giger et al. / Tetrahedron 68 (2012) 7549e7557

can be predicted,19 the Cc-LTA homology model exhibits all the characteristics of a typical Fold Type I protein. The active site is located at the interface of two subunits and the predicted interactions between the consensus residues and the external aldimine and the bound substrate are realized (Fig. 2). Thus, Asp176 and Arg179 are positioned to form salt bridges with the protonated pyridinium ring of the cofactor and the deprotonated substrate carboxylate, respectively. The side chain of the Schiff base-forming lysine, Lys207, is located on the si face of the cofactor, and the His91-Glu96 dyad is found on the re face, within hydrogen bonding distance of the substrate b-hydroxyl group. Asp95, which is adjacent to Glu96, is not conserved in the aldolase family but is located at the substrate entry tunnel of Cc-LTA and could conceivably influence access to the active site.

Fig. 2. Active site of the Cc-LTA homology model. Individual subdomains are colored in white, dark blue, and purple, while conserved residues are shown as green sticks (oxygens, red; nitrogen, blue; phosphorous, orange). The PLP cofactor with bound Lallo-threonine is shown in yellow. (A) The His91-Glu96 dyad packs against the re face of the cofactor and hydrogen bonds with the b-hydroxyl group of the substrate. (B) Surface representation of the enzyme, showing the substrate binding site. The side chain of Asp95 points into this pocket.

2.3. Development of a selection system for threonine aldolase activity In Escherichia coli, glycine can be derived from multiple metabolic precursors (Fig. 3). The principal biosynthetic pathway under normal growth conditions involves conversion of serine to glycine by the enzyme serine hydroxymethyl transferase (glyA).20,21 Glycine is also produced from endogenous threonine via the action of L-allo-threonine aldolase (ltaE)22 and from L-2-amino-3ketobutyrate via 2-amino-3-ketobutyrate lyase (kbl).23 Although

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glyoxylate is potentially another glycine precursor,24 its conversion is likely to be too low to serve as the sole metabolic source of this essential amino acid. To generate a stringent glycine auxotroph for selection experiments with threonine aldolases, we therefore deleted the genes encoding serine hydroxymethyl transferase (DglyA::aadr), L-allo-threonine aldolase (DItaE::kan) and 2-amino3-ketobutyrate lyase (D(tdh-kbl)::cat) from the genome of the E. coli K12 wild-type strain MG1655. Removal of the entire tdh-kbl operon also eliminates the gene for threonine dehydrogenase (tdh), which contributes to threonine degradation.25 The resulting strain, þ3381, does not grow over 10 days in the absence of exogenously added glycine on either solid or liquid M9G media (data not shown). Provision of a gene encoding a functional PLP-dependent retroaldolase, such as Cc-LTA would be expected to restore prototrophy to the þ3381 selection strain, provided that a suitable b-hydroxy-aamino acid is included in the growth medium. Import of the bhydroxy-a-amino acid into the cytosol, followed by retro-aldolasemediated cleavage, would produce an aldehyde and glycine, thus complementing the metabolic deficiency (Fig. 4). To verify this hypothesis, we transformed þ3381 with plasmids encoding either wild type Cc-LTA or GFP as positive and negative controls, respectively. The aldolase gene was placed under the control of the tetracycline-inducible Ptet system to enable systematic variation of intracellular enzyme concentration and hence selection stringency.26 High tetracycline concentrations afford large amounts of enzyme, so even mediocre catalysts should be able to complement the glycine deficiency, whereas at low tetracycline concentrations, relatively little enzyme is produced and highly active aldolases are needed to achieve wild-type levels of growth. Under low stringency conditions (100 ng/mL tetracycline), cells producing the Cc-LTA aldolase reach stationary phase within 24 h in liquid culture supplemented with b-hydroxy-a-amino acids (Fig. 5). The growth rates are comparable to those observed when the control producing GFP is supplemented with 500 mM glycine (data not shown). In the absence of exogenous b-hydroxy-a-amino acid, the cells with intracellular Cc-LTA concentrations still grow, albeit considerably slower. This background activity requires CcLTA, since the GFP control does not detectably grow under the same conditions. The aldolase presumably cannibalizes endogenous pools of threonine and/or serine to produce glycine. At higher stringency (lower tetracycline concentrations), this background growth is significantly reduced. On solid M9G media, for example, no growth is observed over 10 days in the absence of exogenously added b-hydroxy-a-amino acid when Cc-LTA production is induced with 10e50 ng/mL tetracycline. At a given inducer concentration, significantly higher growth rates are observed for Cc-LTA-producing cells supplemented with L-bthreo-phenylserine than with L-threonine (Fig. 5). Assuming that both amino acids are taken up by the cells with similar efficiency, this result suggests that the aromatic amino acid is a better substrate for the enzyme. Consistent with this interpretation, growth on exogenously added L-threonine becomes steadily less efficient as the CcLTA concentration is reduced by lowering the tetracycline concentration from 100 ng/mL to 10 ng/mL. With L-b-threo-phenylserine, in contrast, a significant decrease in growth rate is only observed under the most stringent selection conditions, i.e., at 10 ng/ml tetracycline. For both substrates, growth rates also decrease as the concentration of the b-hydroxy-a-amino acid is lowered (Fig. 6). 2.4. Investigation of Cc-LTA using the glycine auxotrophic strain The possibility of controlling cell growth and selection stringency by independently varying three parametersdsubstrate structure, substrate concentration and tetracycline concentrationdmakes this system a versatile tool for probing the

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Fig. 3. Glycine metabolism in wild-type and reprogrammed E. coli cells. Biosynthesis of glycine and serine is tightly coupled with conversion of tetrahydrofolate (THF) to methylenetetrahydrofolate, the metabolic cofactor that delivers C1 moieties to numerous pathways producing essential building-blocks, including purine nucleotides of RNA and DNA, thymidylate for DNA, methionine and formylmethionine for proteins, as well as the cofactors S-adenosylmethionine, glutathione, biotin, pantoate, riboflavin and tetrahydrofolate itself. Deletion of glyA, the gene for serine hydroxymethyl transferase, does not block the endogenous production of glycine nor of methylenetetrahydrofolate, owing to the presence of three salvage pathways in wild-type E. coli K12. The first catalyzes the decarboxylative oxidation of glycine to ammonia, carbon dioxide, and methylenetetrahydrofolate through the action of glycine decarboxylase, the multi enzymatic complex specified by the gcvTHP operon (glycine cleavage system). The second, specified by the kbl-tdh operon, enables the conversion of threonine into glycine and acetyl-coenzyme A (AcSCoA), through the action of 2-amino-3-ketobutyrate lyase and threonine 3-dehydrogenase. The third, specified by the ltaE gene for L-allo-threonine aldolase, enables the retro-aldolization of threonine into glycine and acetaldehyde. Deleting ltaE and the kbl-tdh operon in addition to glyA (red arrows) results in a genetic background conferring the nutritional requirement for an exogenous source of glycine or a precursor thereof. This growth requirement provides a nutritional screen for selecting plasmid-encoded aldolases (green arrow) catalyzing the retro-aldol reaction of stereochemically diverse b-hydroxy-a-amino acids. Red arrows indicate reactions eliminated by gene deletions and the green arrow shows the enzymatic step specified by plasmid-borne mutant genes.

Fig. 4. In vivo complementation strategy for threonine aldolase activity. The gene of a potential aldolase, such as Cc-LTA (red arrow) is expressed from the pMG-Ptet plasmid in the glycine auxotroph using the tetracycline-inducible Ptet system, which provides graded and homogeneous transcriptional control of catalyst production. If the enzyme is active as an aldolase, it will convert the supplied b-hydroxy-a-amino acid to an aldehyde and glycine and thus complement the glycine deficiency.

properties of threonine aldolases by combinatorial mutagenesis. In proof-of-concept experiments, we targeted Asp176, the conserved residue that interacts with the cofactor pyridinium nitrogen; the catalytic His91-Glu96 dyad; and Asp95, which is not conserved but could influence substrate binding. A combinatorial Cc-LTA library, constructed from randomized oligonucleotides containing NNS codons at the four target sites,

was used to transform electrocompetent þ3381 cells. Transformation efficiencies of >106 colony forming units per microgram DNA ensured adequate coverage of the 1.6105 theoretically possible protein variants. The recovered cells were washed to remove threonine and glycine, and the library was plated on agar plates containing either 200 mM L-threonine or 200 mM L-allo-threonine at two different inducer concentrations, 10 and 50 ng/mL tetracycline, to adjust selection stringency. After 6 days on plates supplemented with L-threonine and 50 ng/mL tetracycline, only 3.5% of the plated transformants afforded single colonies; under the more stringent selection conditions, the number of complementing clones decreased fivefold to 0.7%. In contrast, 16e18% of the library grew on plates supplemented with L-allo-threonine after 4 days, irrespective of the inducer concentration. For each set of conditions, fifteen plasmids from representative functional clones were isolated and sequenced to determine the amino acid preferences at the target sites (Supplementary data Table S2 and S3). Interestingly, Asp176 and the His91-Glu96 dyad were invariant in all clones obtained from the selection experiments with L-threonine as the glycine precursor. Asp95 in the substrate entry tunnel exhibited greater tolerance to substitution. Although the wild-type aspartate was retained in the majority of recovered variants (17/30), it can also be replaced by bulky hydrophobic residues, such as tryptophan (8/30), methionine (3/30), tyrosine (1/30) and leucine (1/30). The Cc-LTA variants obtained in the selection experiments with L-allo-threonine exhibited a larger range of substitutions, as expected from the higher complementation frequency. Only His91 was absolutely conserved, consistent with its postulated role as the catalytic base. Asp95 was again replaceable by a variety of amino acids, including cysteine (1/30), asparagine (1/30), tyrosine (1/30), histidine (1/30), and leucine (3/30). Glu96 was more

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Fig. 5. Tetracycline-dependent growth of the E. coli glycine auxotroph +3381. Cc-LTA producing cells (filled symbols) were grown at 30  C in 4 mL test tubes in minimal medium containing either (A) 100 mM L-b-threo-phenylserine or (B) 200 mM L-threonine. Gene expression was induced with a range of tetracycline concentrations: red, 100 ng/mL; blue, 50 ng/mL; green, 25 ng/mL; black, 10 ng/mL. Cells producing GPF instead of Cc-LTA (empty symbols) were grown under the same conditions and served as negative controls.

Fig. 6. Substrate-dependent growth of the E. coli glycine auxotroph +3381. Cc-LTA producing cells (filled symbols) were grown at 30  C in 4 mL test tubes in minimal medium containing 50 ng/mL tetracycline and (A) 50, 100, 250 or 500 mM L-b-threo-phenylserine (black, green, blue, and red, respectively) or (B) 100, 200, 500 or 1000 mM (black, green, blue, and red, respectively) L-threonine. Cells producing GPF instead of Cc-LTA (empty symbols) were grown under the same conditions and served as negative controls.

conserved but can be substituted by glycine (1/30), serine (1/30), and threonine (1/30). Even a conservative mutation of Asp176 to glutamate (2/30) was observed. For comparison, the sequences of ten library variants that had not been subject to selection confirmed that the starting library was diverse and contained neither off-target mutations nor frame shifts (Supplementary data Table S1).

Table 2 Kinetic parameters of PLP-dependent aldolases

a

Cc-LTA

Substrate

kcat (s1)

Km (mM)

kcat/Km (M1 s1)

L-Threonine

3.3 20 67.4 580 1.3103 2.3103 1.0 3.4 3.8

13.6 0.69 0.035 29 22 1.3 2.9 0.22 0.12

2.4102 2.9104 1.9106 2.0104 5.9104 1.8106 3.4102 1.5104 3.2104

L-allo-Threonine L-

2.5. Kinetic characterization of Cc-LTA variants

Pp-LPSb

Wild-type Cc-LTA and all the single and double mutants identified in the selection experiments were produced and purified for kinetic characterization. Retro-aldolase activity with L-threonine, Lallo-threonine and L-b-threo-phenylserine was measured for each enzyme. Table 2 lists the steady-state parameters for wild-type Cc-LTA and two previously characterized threonine and b-phenylserine aldolases. Based on its substrate preferences (L-b-threo-phenylserine>>L-allo-threonine>>L-threonine), Cc-LTA should evidently be classified as a b-L-phenylserine aldolase (LPS). A kcat/Km value in excess of 106 M1 s1 for the aromatic substrate is particularly notable in this context. Although substrate promiscuity is not unusual for PLP-dependent serine hydroxymethyl transferases and aldolases,6,7,29,30 the 380-fold lower Km value for L-b-threo-phenylserine compared to L-threonine makes Cc-LTA considerably more selective than the previously characterized P. putida b-phenylserine aldolase.27

Ec-LTAb

b-threo-Phenylserine

L-Threonine L-allo-Threonine L-

b-threo-Phenylserine

L-Threonine L-allo-Threonine L-

b-threo-Phenylserine

Kinetic parameters were determined at 30  C using a continuous assay in 50 mM PBS buffer, pH 8.0. b Representative kinetic parameters for a previously described L-phenylserine aldolase from P. putida (Ref. 27) and an L-threonine aldolase from E. coli (Ref. 28). a

Substitution of Asp176 with glutamate is highly deleterious (Table 3). Previous analysis of Fold Type I enzymes identified this residue, which interacts with the pyridinium nitrogen of PLP, as a defining feature of this enzyme superfamily,12 and it is conserved across the entire family of threonine aldolases (Table 1). Nevertheless, the D176E mutation was found twice in our moderately stringent selection experiments with L-allo-threonine. Steady-state measurements confirm that this Cc-LTA variant is active, but the extra methylene group of glutamate is poorly accommodated at the

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Table 3 MichaeliseMenten parameters of selected Cc-LTA single mutantsa Clone

Cc-LTA D176E D95L D95C D95M D95Y D95W

L-Threonine

L-allo-Threonine

L-

b-threo-Phenylserine

kcat (s1)

Km (mM)

kcat/Km (M1 s1)

kcat (s1)

Km (mM)

kcat/Km (M1 s1)

kcat (s1)

Km (mM)

kcat/Km (M1 s1)

3.3 d 3.8 3.0 3.6 3.5 9.6

13.6 d 53 44 50 63 44

240 <1 72 68 72 56 218

20 d 22 19 nm 20 19

0.69 d 12 11 nm 2.7 2.5

2.93104 <1 1.83103 1.73103 nm 7.43103 7.63103

67 0.1 23 22 21 14 17

0.035 0.24 0.19 0.17 0.58 0.31 0.78

1.9106 4.2102 1.2105 1.3105 3.6104 4.5104 2.2104

nm: not measured with this substrate. a Kinetic parameters were determined at 30  C using a continuous assay in 50 mM PBS buffer, pH 8.0. Table 4 MichaeliseMenten parameters of selected Cc-LTA double mutantsa Clone

L-Threonine

kcat (s1) Cc-LTA D95Y, E96T D95H, E96G D95N, E96S a

3.3 d 0.6 0.2

L-allo-Threonine

Km (mM) 13.6 d 13 32

kcat/Km (M1 s1) 240 1.6 46 6.3

kcat (s1) 20 3.0 8.6 0.2

L-

Km (mM) 0.69 4.4 2.9 7.1

kcat/Km (M1 s1) 4

2.9310 6.83102 3.03103 28

b-threo-Phenylserine

kcat (s1)

Km (mM)

kcat/Km (M1 s1)

67 2.8 6.8 5.5

0.035 0.027 0.071 0.065

1.9106 1.0105 9.6104 8.5104

Kinetic parameters were determined at 30  C using a continuous assay in 50 mM PBS buffer, pH 8.0.

active site and causes a >102-fold drop in kcat and a >103-fold drop in kcat/Km. That such a weakly active variant could be isolated from the combinatorial libraries highlights the large dynamic range of the engineered selection system. Of the four randomized sites, residue 95, which is not conserved in the threonine aldolase enzyme family, is clearly the most tolerant to substitution. Five of the variants containing single mutations had Asp95 replaced by another, typically hydrophobic amino acid. These substitutions increase the Km values for all of the substrates (Table 3), consistent with the side chain of this residue projecting into the substrate binding pocket. The most sterically demanding substrate, L-b-threo-phenylserine, is particularly sensitive to these substitutions, as evidenced by the additional deleterious effect on kcat (Table 3). In contrast, the catalytic rate constant for the smaller substrates is either unchanged (L-allo-threonine) or can even increase, as seen for the D95W variant with L-threonine, compensating for the increase in Km. The latter observation presumably explains the frequent occurrence of the D95W mutation (>25%) in the selection experiments with L-threonine. Mutations of Glu96, the hydrogen bonding partner of His91,16 were only found in combination with mutations of adjacent Asp95. All three variants are less active than wild-type Cc-LTA, with the reduction in catalytic efficiency ranging between 5 and 1000fold for the different substrates (Table 4). In contrast to the Asp95 single mutants, the double mutants exhibit more significantly impaired kcat values. For example, D95Y/E96T Cc-LTA has a 5-fold lower kcat than the D95Y variant with L-b-threo-phenylserine. Nevertheless, because Km also decreases 11-fold, the double mutant has a 2.5-fold higher kcat/Km with this substrate. The fact that this same variant is essentially inactive with L-threonine suggests that the origins of these effects are likely to be complex, and additional work will be needed to tease apart how the subtle structural changes at the active site differentially influence the rate and binding constants for the different substrates. 3. Conclusions Genetic selection is a powerful tool in mechanistic enzymology and protein design.9,10 Coupling enzyme activity to cell growth makes the analysis of very large mutant libraries possible. Since only cells producing a functional catalyst survive, more than >107 variants can be easily analyzed in parallel in a single experiment.

Nevertheless, living organisms are complex and development of a tight selection system is often challenging, limiting widespread application of this approach. Our results show that simultaneous inactivation of four essential Escherichia coli genes in glycine, serine and threonine metabolism affords a versatile in vivo selection strain for PLP-dependent Lthreonine aldolases. The resulting cell line is unable to grow in the absence of exogenously added glycine unless a functional enzyme able to cleave b-hydroxy-a-amino acids is provided. Further, regulable selection stringency was achieved by simple adjustments to intracellular aldolase concentration, b-hydroxy-a-amino acid structure, and/or the extracellular substrate concentration. Transcriptional control of gene expression is a versatile strategy for regulating the stringency of selection experiments.31 As in the case of the Ptet system that we employed, growth-limiting aldolase concentrations can be matched to activity levels needed to overcome selection hurdles. For example, weakly active catalysts like the D176E Cc-LTA variant can be isolated from large protein libraries when produced at very high levels. In contrast, highly active catalysts like wild-type Cc-LTA confer a growth advantage to the host strain even at very low concentration. Such control over catalyst concentration should facilitate the directed evolution of weak or moderately active aldolases. Selection pressure can be steadily increased in each round of mutagenesis simply by reducing the catalyst concentration to ensure that only cells harboring the most efficient variants survive. If necessary, degradation tags directing the aldolase to cellular proteases may be used to achieve even higher selection stringencies.26,32 Growth rates of the engineered glycine auxotroph are also sensitive to the choice of substrate. Many threonine aldolases, like CcLTA, are promiscuous. We find that complementation efficiency roughly correlates with the specific activities of the b-hydroxy-aamino acid used as a glycine precursor. Thus, roughly 10-times more Cc-LTA variants were recovered on plates supplemented with L-allothreonine rather than L-threonine, reflecting the ca. 100-fold higher kcat/Km value for this substrate. Moreover, the more active substrate enabled selection of variants spanning a 100-fold wider activity range. Although we only tested three different substrates, the choice of b-hydroxy-a-amino acid is only limited by cellular uptake and turnover efficiency of the target enzyme.33 In principle, both Dand L-configured substrates, threo and erythro stereochemistries, and a wide variety of side chains could be utilized in conjunction

L. Giger et al. / Tetrahedron 68 (2012) 7549e7557

with an appropriate aldolase. The ability to control selection stringency by adjusting both catalyst and substrate concentration is an appealing feature of this system. Accordingly, selection pressure may be adjusted to accommodate the large variations in substrate affinity characteristic of this enzyme class. Threonine aldolases have significant potential as catalysts for the synthesis of diverse b-hydroxy-a-amino acids.5e8,29,34e37 A better understanding of their catalytic machinery may aid efforts to reengineer substrate specificity and thus expand the scope of donor and acceptor substrates. Analysis of multiple sequence alignments of 95 unique L-threonine aldolases in conjunction with available structural data has highlighted several highly conserved features in the aldolase active site. These features include an aspartate involved in binding the protonated cofactor and a Glu-His dyad that might facilitate proton abstraction during catalysis. Our selection experiments confirm the importance of the corresponding residues in Cc-LTA. Even conservative substitution of Asp176 with glutamate reduced catalytic efficiency >104-fold, and no viable replacements for His91 were found. If the histidine were only important for binding PLP via p-stacking, other aromatic amino acids, such as tyrosine or phenylalanine would have been expected to be reasonable replacements. In fact, such residues appear in structurally homologous PLP-dependent enzymes that promote other reaction types.12 The selection results also indicate that Glu96, while not essential, may modulate histidine reactivity. The consequences of mutating this residue were less deleterious than substitution of the analogous aspartate in the serine protease catalytic triad.38,39 Still, substitutions arose only in conjunction with mutations of the adjacent Asp95, suggesting that subtle structural changes at the active site were concomitantly required. Notably, permissible replacements are small (glycine) or hydrophilic (serine, threonine). It is conceivable that a water molecule might bind within the cavity generated by these mutations and, by providing a hydrogen bond to His91, somewhat mitigate the effects of removing the carboxylate. In summary, a versatile selection system for PLP-dependent aldolases has been created and applied to the study of selected amino acids lining the active site of a promiscuous b-hydroxy-a-amino acid aldolase. Such a selection system opens up new possibilities for exploring enzyme structure-function relationships and for directed evolution of improved biocatalysts for stereoselective synthesis of pharmaceutically important b-hydroxyamino acids.

4. Materials and methods 4.1. Multiple sequence alignment The UniProt reference cluster database of the European Bioinformatics Institute (EBI) (http://www.ebi.ac.uk/uniref/) was searched for threonine aldolases.40,41 Sequence pairs with higher than 50% mutual sequence identity were excluded to minimize redundant information in the alignment, which reduced the initial 1408 cluster members to 216 putative threonine aldolases (90% sequence identity yields 936 variants). The extracted sequences were subsequently aligned using the UniProt online interface. Following removal of predicted D-threonine aldolases, alanine racemases, and tryptophan synthases, as well as sequences containing fewer than 150 or more than 450 bp, 168 independent threonine aldolase sequences remained (full alignment is provided as Supplementary data).

4.2. Homology model The Swiss-Model workspace42 was used to generate a homology model of the Cc-LTA tetramer using published protocols.19

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4.3. DNA manipulations Molecular cloning was performed according to standard procedures.43 Restriction endonucleases and T4 DNA ligase were obtained from New England Biolabs (Ipswich, MA). Oligonucleotides were custom synthesized by Microsynth AG (Balgach, Switzerland). Polymerase chain reactions (PCRs) were performed using Phusion polymerase from Finnzymes OY (Espoo, Finland). All PCRderived segments in the constructed plasmids were confirmed by DNA sequencing on a 3100-Avant Genetic Analyzer (Applied Biosystems, Foster City, CA) by chain termination chemistry44 with the BigDye Terminator Cycle Sequencing Kit from the same company. 4.4. Construction of an E. coli glycine auxotroph The glycine auxotroph, E. coli strain þ3381, has the genotype

DglyA::aadr, D(tdh-kbl) and DltaE::kan. It was derived from wild

type E. coli K12 strain MG1655 by serial P1 transductions of the three deletion alleles DglyA::aad, D(kbl-tdh)::cat and DltaE::kan. Knockouts of the glyA gene and of the kbl-tdh operon were generated in a recB recC sbcB background by homologous recombination of the chromosome with a linear DNA construct consisting of an antibiotic resistance cassette flanked by 500 base pair DNA stretches homologous to the upstream and downstream sequences of the genomic locus to be deleted according to a published procedure.45 The in-frame deletion of the ltaE coding sequence was obtained by homologous recombination of PCR product mediated by the phage l recombinase following a published procedure.46 The intermediate strains þ1184 (DglyA::aad), þ3174 (DglyA::aad D(kbltdh)), þ3367 (DglyA::aad DltaE::kan) and þ3381 were checked for growth in mineral medium glucose without added glycine. The strains þ1184 and þ3367 were able to grow in the absence of glycine, although the growth onset was delayed by a 12 h-long lag phase. No growth was observed with þ3174 or þ3381 strains even after prolonged incubation (10 days). 4.5. Dynamic range of the glycine auxotroph Plasmids encoding Cc-LTA or GFP were used to transform Ca2þcompetent þ3381 cells. þ3381 cells transformed with the library plasmid were subjected to selection on M9G agar plates containing 20% glucose. The latter are based on M9c minimal agar plates47 containing 150 mg/mL sodium ampicillin, 150 mM pyridoxine, and 10e50 ng/mL tetracycline, but lacking glycine. In contrast to medium for chorismate mutase selections,48 thiamine, aromix (4hydroxybenzoic acid, 4-aminobenzoic acid, 2,3-dihydroxybenzoic acid), and tryptophan were not added. As a positive control, M9G plates supplemented with glycine (0.2 mM) were used. Alternatively, single colonies were grown overnight in 5 mL LB medium. The next day, the cells were washed three times by centrifugation and resuspension in 4 ml M9G salts43 at 4  C to remove residual Lthreonine and glycine. A 40 mL aliquot of resuspended cells was diluted to a final volume of 4 mL M9G salts in test tubes, tetracycline and substrate were added, and the mixtures were incubated at 30  C for 48 h. Cell growth was monitored at 600 nm (Figs. 4e6). 4.6. Library construction Previously described pMG-Ptet-GFP served as the library vector.26 The GFP gene was replaced by the alanine racemase gene from Cochliobolus carbonum using NdeI and XhoI restriction sites to provide a library vector (pMG-Ptet) containing a stuffer fragment that is considerably larger (>1500 bp) than the 1051 bp Cc-LTA gene. The large insert facilitated discrimination between singly cut and fully digested vector. The Cc-LTA gene was initially amplified from genomic DNA and cloned into pMG-Ptet by standard PCR

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techniques using flanking primers Cc-LTA3f (50 -TGATAACATATGACCCAGACCGCGC-30 , NdeI restriction site underlined) and CcLTA3r (50 -TTATCACTCGAGAGCCACTCGCTTC-30 , XhoI restriction site underlined). The full-length gene was digested in parallel with the pMG-Ptet vector using NdeI and XhoI as endonucleases according to the manufacturer’s protocol. After agarose gel purification, acceptor and insert were mixed in a 1:3 stoichiometric ratio and ligated with T4 DNA ligase. The ligation mix was incubated overnight and purified by chloroform/phenol extraction; the aqueous phase was washed three times using YM-30 columns from Microcon (Bedford, MA, USA).49 Libraries were constructed by standard overlap extension PCR50 using pMG-Ptet-Cc-LTA as a template. The codons for His91, Asp95 and Glu95 were simultaneously mutagenized using the degenerate NNS codon, where N represents an equimolar mixture of all four nucleotides and S an equimolar mixture of C and G. The library primer Cc-LTA_lib1af (50 -GCCCACGAGCACGCCNNSATCTGCACGNNSNNSACCGGCGCGCCCG-30 ) and the Cc-LTA3r flanking primer were used to produce the first PCR fragment. A second fragment was generated using the Cc-LTA_lib1br (50 GGCGTGCTCGTGGGC-30 ) and Cc-LTA3r primers. After purification on a 2% agarose gel, the two fragments were assembled into the full-length gene with the Cc-LTA3f and Cc-LTA3r primers. The codon for Asp176 was subsequently randomized utilizing the fulllength Cc-LTA gene library as a template and LTA_lib1bf (50 GCTACGGCGTCCACCTGNNSGGCGCGCGGCTGG-30 ) and Cc-LTA3r (50 -TTATCACTCGAGAGCCACTCGCTTC-30 ) as primers as described above. The reassembled gene was digested with NdeI and XhoI and ligated into the pMG-Ptet acceptor vector. The desalted ligation mixture was added to 50 mL precooled electrocompetent þ3381 cells (OD600>200) and transformed by electroporation. The cells were immediately recovered in 25 mL SOC medium43 and incubated for 45 min at 30  C. Library size was determined by serial dilution on LB plates supplemented with sodium ampicillin (150 mg/ml), and representative clones were sequenced to verify library quality. The bulk of the culture was also supplemented with sodium ampicillin (150 mg/ml) and further incubated at 30  C overnight for preparation of glycerol stocks (library storage). 4.7. In vivo selection experiments The pool of þ3381 cells containing the gene library was grown in LB medium containing sodium ampicillin (150 mg/ml). Cells were washed three times with liquid M9c medium to remove any residual amino acids. Serial dilutions were plated in parallel onto minimal M9G plates lacking glycine but containing 10 ng/mL or 50 ng/mL tetracycline and 0.2 mM L-threonine or 0.2 mM L-allothreonine. To determine the complementation rate, aliquots from each dilution were also plated on M9G plates containing 0.2 mM glycine. The plates were incubated at 30  C for 3e5 days. Colonies were picked from selective plates based on colony size and plate (substrate and tetracycline concentration) for sequence analysis and plasmid isolation. 4.8. Protein production Plasmids were isolated from individual clones, purified using the NucleoSpin Extract II kit (MachereyeNagel, Germany), and used to retransform þ3381 cells. Overnight cultures were used to inoculate 500 ml LB medium (supplemented with 150 mg/ml sodium ampicillin) to an OD600 of 0.7 at 37  C with shaking at 180 rpm. Twenty hours after induction with 0.5 mg/ml tetracycline, cells were harvested by centrifugation (4000g, 20 min, 4  C) and resuspended in 3 pellet volume phosphate buffer (50 mM sodium phosphate, 300 mM NaCl, pH 8) containing 10 mM imidazole. Lysozyme (1 mg/mL) was added to the cell suspension and incubated

for 30 min on ice before cell lysis was initiated by sonication. Before centrifugation (>10,000 x g, 20 min, 4  C) a mixture of DNase (5 mg/ ml) and RNase (5 mg/ml) was added and incubated 15 min on ice. 1 mL Ni-NTA agarose (QIAGEN) was added to the soluble fraction and incubated in a gravity flow column for 5 min at room temperature. The beads were washed with phosphate buffer (pH 8) containing 20 mM and 40 mM imidazole, and the protein was eluted with about 2 mL phosphate buffer (pH 8) containing 250 mM imidazole. The eluate was dialyzed twice against 1 L 50 mM sodium phosphate buffer (pH 8) at 4  C and filtered through a 0.22 mm sterile filter before storing at 4  C. 4.9. Activity assay for retro-aldol cleavage The conversion of L-b-threo-phenylserine to benzaldehyde and glycine was monitored by UV spectroscopy (D3 279nm¼1400 M1 cm1).51e53 Reactions were conducted at 30  C in 1 mL 50 mM sodium phosphate buffer (pH 8), containing 5 mM enzyme, 10 mM PLP and between 0.1 mM and 6 mM racemic D,L-bthreo-phenylserine in a 1 cm path length cuvette. In the case of L-threonine or L-allo-threonine the enzymatic cleavage was assayed by coupling the reaction to the NADH-dependent reduction of acetaldehyde by yeast alcohol dehydrogenase.54 The decrease in NADH concentration was followed by UV spectroscopy (D3 340nm¼6200 M1 cm1). 200 mM NADH, 30 units/mL yeast alcohol dehydrogenase, 5 mM enzyme, 10 mM PLP, between 2 and 120 mM L-threonine or 0.5 and 15 mM L-allo-threonine were mixed in 1 mL 50 mM sodium phosphate buffer (pH 8), at 30  C. Acknowledgements rie Delmas for expert technical assistance with We thank Vale strain constructions. The financial support of the Schweizerischer Nationalfonds and the Stipendienfonds der Schweizerischen Chemischen Industrie is gratefully acknowledged. Supplementary data Supplementary data associated with this article can be found in the online version, at doi:10.1016/j.tet.2012.05.097. References and notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

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