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α-hydroxylated aldehydes (C2-C3) led to the best TK activities. As regards the TK-catalyzed reaction stereoselectivity, the newly-formed asymmetric c...
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Evolved Thermostable Transketolase for Stereoselective Two-Carbon Elongation of Non-Phosphorylated Aldoses to Naturally Rare Ketoses Marion Lorillière, Romain Dumoulin, Melanie L'enfant, Agnes Rambourdin, Vincent Thery, Lionel NAUTON, Wolf-Dieter Fessner, Franck Charmantray, and Laurence Hecquet ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.9b01339 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 18, 2019

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ACS Catalysis

Evolved Thermostable Transketolase for Stereoselective TwoCarbon Elongation of Non-Phosphorylated Aldoses to Naturally Rare Ketoses Marion Lorillière,† Romain Dumoulin,† Mélanie L’enfant,† Agnès Rambourdin,† Vincent Thery,† Lionel Nauton,† Wolf-Dieter Fessner,‡ Franck Charmantray,*,† Laurence Hecquet*,† † Université Clermont Auvergne, CNRS, SIGMA Clermont, Institut de Chimie de Clermont-Ferrand (ICCF) F-63000 Clermont-Ferrand, France ‡ Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Alarich-WeissStrasse 4, 64287 Darmstadt, Germany

ABSTRACT: We propose an ecofriendly, efficient, stereoselective procedure for the two-carbon elongation of nonphosphorylated aldoses (C4–C6) to the corresponding Cn+2 ketoses (C6–C8) in one step, using hydroxypyruvate (HPA) as a ketol donor substrate and an evolved thermostable transketolase from Geobacillus stearothermophilus (TKgst) as biocatalyst. Simultaneous site saturation mutagenesis (SSM) at two or three key positions in the TKgst active site yielded efficient variants, L382F/F435Y, R521Y/S385/H462N and R521V/S385D/H462S, with increased activity compared to wildtype TKgst for conversion of two tetroses (D-threose, L-erythrose), two pentoses (D-xylose, D-ribose) and two hexoses (Dallose, D-glucose), respectively. These six Cn aldoses as acceptor and HPA as donor substrates were transformed by the TKgst variants at 60°C with practically complete conversion. The corresponding Cn+2 ketoses, including two hexuloses (Dtagatose, L-psicose), two heptuloses (D-altro-heptulose, D-ido-heptulose) and two octuloses (D-glycero-D-ido-octulose, Dglycero-D-altro-octulose) are naturally rare compounds with important biological functions, which were obtained with high diastereoselectivity. KEYWORDS : biocatalysis, asymmetric synthesis, C-C bond formation, transketolase, ketoses, in vitro evolution

chain in monosaccharides broadens their synthetic scope and stereo configurational diversity.3 Typical synthetic routes for higher-carbon sugars involve Short-chain carbohydrates are valuable chiral homologation of lower-carbon sugars requiring the building blocks in the synthetic laboratory while a introduction of new stereogenic centers in a controlled number of higher-carbon sugars found in nature in manner with tedious protection/deprotection very low amounts, so-called rare sugars, have 4 manipulations. important biological functions.1 With the exception of As an attractive alternative to chemical chain D-fructose, all ketoses are defined as rare sugars that elongation, enzymatic reactions have emerged offering offer enormous potential for applications in functional several advantages, such as high substrate and stereo food, pharmaceutical, medicinal and synthetic specificity, avoidance of laborious chemistry.2 Because most of the rare ketoses are not protection/deprotection sequences, and mild energyreadily available, this fundamental class of efficient reaction conditions.5 While the toolbox of carbohydrates still requires the development of biocatalysis encompasses a large repertoire of efficient synthetic processes. In carbohydrate functionally diverse and robust enzymes from natural chemistry, the synthesis of chain-elongated sugars is a sources, their catalytic activity and selectivity for central research challenge for more than a century, in specific targets of human interest must be improved particular because adjusting the length of the carbon via in vitro evolution or rational, structure-based ACS Paragon Plus Environment

INTRODUCTION

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design, or a combination of both engineering strategies, to become competitive with the productivity of traditional methods in chemocatalysis.6 Aldose-ketose isomerization by enzymatic way is the most important method for ketose preparation, even though the reaction equilibrium is most often unfavorable for ketose formation. However, these processes suffer from low yields, expensive starting materials, coupling with other enzymes or need a complicated isomer separation step.7 For chain elongation of monosaccharides, enzymes catalyzing a stereospecific C-C bond formation such as aldolases and transketolases (TK) are most popular. In vivo, these enzymes are involved in metabolic pathways where they interconvert phosphorylated ketoses and aldoses in reversible reactions. In vitro application of these enzymes for non-phosphorylated ketose synthesis, particularly with extended carbon chains, often requires multi-enzymatic strategies and the improvement of enzyme activities toward non natural substrates by protein engineering. Aldolases catalyze asymmetric reactions from an enolizable carbonyl compound and an aldehyde acceptor, enabling three-carbon chain elongation.8 However, for the synthesis of non-phosphorylated long carbon chain rare ketoses differently configurated on C3 and C4 aldolase-catalyzed reactions most often require phosphorylated substrates and additional enzymes to liberate free ketoses as well as mutagenesis approaches to improve enzyme efficiency.8a,9 TK (EC 2.2.1.1), belonging to the thiamine diphosphate-dependent enzyme family, catalyzes a two-carbon chain elongation by transferring an αhydroxy carbonyl (ketol) group from a donor to an aldehyde acceptor, thereby producing the Cn+2 ketose (Scheme 1). In nature, TK is involved in the pentose phosphate metabolic pathway where this enzyme transfers a ketol group to reversibly equilibrate ketoses and aldoses carrying a terminal phosphate ester group. Previous in vitro studies showed that nonphosphorylated aldose compounds can also be used as TK substrates leading to the corresponding free ketoses in one step. Particularly, release of carbon dioxide from hydroxypyruvate (HPA) as donor kinetically favours the reaction, thus making TK a powerful tool for the asymmetric synthesis of ketoses and related acyloin compounds (Scheme 1).10

Page 2 of 19 O

HO

OH CO2H

+

R O Cn (4-6)

Donor HPA

Acceptor (2R)-hydroxylated aldehyde

TK ThDP, Mg2+

O

OH

HO

R

+

CO2

OH Cn+2 (6-8) Product (3S,4R) ketose

Scheme 1. Irreversible reaction catalyzed by TKs in the presence of hydroxypyruvate (HPA) as donor substrate with (2R)-hydroxyaldehydes as acceptor substrates.

The studies of TK acceptor substrate specificity revealed that non-phosphorylated short carbon chain -hydroxylated aldehydes (C2-C3) led to the best TK activities. As regards the TK-catalyzed reaction stereoselectivity, the newly-formed asymmetric carbon of the Cn+2 ketose product exhibits an (S) configuration and the enzyme is highly enantioselective with regard to the C2 configuration of chiral α-hydroxyaldehydes, and preferentially accepts the (2R)-epimer, leading to (3S, 4R) ketoses. For biocatalytic applications, mesophilic TKs from Saccharomyces cerevisiae (TKsce) and from Escherichia coli (TKeco) have been largely used11 and optimized by mutagenesis.12,13 We recently identified the first thermostable TK from Geobacillus stearothermophilus (TKgst), offering significantly improved stability at high temperature and more robustness toward unusual reaction conditions.14-16 We were able to improve and to broaden the TKgst substrate specificity and enantiospecificity using in vitro evolution toward (2S)hydroxylaldehydes with short carbon chains,17 aliphatic and aromatic aldehydes18,19 and also toward HPA analogs as new donor substrates.20 A major issue to be addressed is to improve TK activity toward non-phosphorylated long-chain aldoses as acceptors for obtaining the corresponding Cn+2 ketoses. Only a few studies with this aim have been described in the literature so far, notably the synthesis of L-gluco-heptulose from L-arabinose using a TKeco variant12d but in most cases the ketose products were not isolated and characterized.13 The present study focuses on the development of an original, efficient, stereoselective two-carbon elongation procedure from non-phosphorylated differently configurated aldoses (C4-C6) and HPA as a unique donor substrate for obtaining the corresponding Cn+2 ketoses at preparative scale in one step. The improvement of TKgst activity by in vitro evolution toward long carbon chain nonphosphorylated aldoses was performed using a semi-

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ACS Catalysis

rational mutagenesis strategy applied simultaneously on two or three key positions to favor the binding of the targeted aldoses. Non-phosphorylated commercially available aldoses of increased carbon chain length (C4–C6) were used as putative TK acceptor substrates: the tetroses D-threose 1 and its (3S)-epimer L-erythrose 2, the pentoses D-ribose 3 and its (3S)epimer D-xylose 4, and the hexoses D-allose 5 and its (3S)-epimer D-glucose 6. The best identified TKgst variants were used in a one-pot, one-step procedure in the presence of HPA and selected aldoses to obtain the corresponding naturally rare and valuable Cn+2 ketoses 7-12 (Table 1). Table 1. TKgst-catalyzed reaction from Cn aldoses 1-6 and HPA to Cn+2 ketoses Aldose acceptors CHO-R

Ketose products Cn

CH2OH-CO-CHOH(S)-R

OH

O

(R)

(S)

4

OH

HO

O

(S)

OH

4

HO

O OH

(R)

5

HO

O

OH

(R)

5

HO

(R) (R)

6

HO

(R) (R)

OH OH

D-glucose 6

OH

7

OH

(S)

(R)

(R)

(R)

(R)

OH

8

D-glycero-D-altro-octulose 11

OH OH (S)

OH (R)

OH OH OH

D-allose 5

(R)

(S)

OH OH

O OH

OH OH

O

7

D-ido-heptulose 10

OH OH (R)

(R)

OH

D-xylose 4

(R)

OH

(R)

OH

OH (S)

OH

O

OH

(R)

D-altro-heptulose 9

OH OH (S)

(R)

OH

D-ribose 3

(R)

6

OH

OH (S)

OH

O

(S)

L-psicose 8

OH OH (R)

(S)

OH OH

L-erythrose 2

(R)

6

OH

OH (S)

OH

O

(R)

D-tagatose 7

OH (S)

(S)

OH OH

D-threose 1

O

(S)

OH

O

Cn+2

OH

OH OH

O OH

6

HO

(S)

(R)

(S)

(R)

(R)

OH

8

OH OH OH

D-glycero-D-ido-octulose 12

The ketoses 7-12 have important biological functions. D-tagatose 7 (C6) displays antidiabetic properties and is used in the food industry as a hypocaloric sweetener.21,22,L-Psicose 8 (C6) is used for the synthesis of L-fructose, precursor of an inhibitor of glucosidases.23,24 D-altro-heptulose (D-sedoheptulose) 9 (C7) is a marker of sugar metabolism disorders such as cystinose.25,26 D-ido-heptulose 10 has been reported to be a precursor of valiolamine and N-substituted derivatives, glucosidase inhibitors useful for the

treatment of hyperglycemic symptoms.27 D-glycero-Daltro-octulose 11 (C8) has been identified in plant, Spinacia oleracea28 and its (5S)-epimer D-glycero-D-idooctulose 12, which is very abundant in Craterostigma plantagineum, has been described as a plant antioxidant involved in desiccation tolerance28 and could be a potential reactive oxygen species (ROS) scavenger with applications in nutrition and healthcare.29

RESULTS AND DISCUSSION Construction of TKgst variant libraries. TKs being involved in the pentose phosphate pathway, active sites of wild-type enzymes are designed for the binding of phosphorylated aldoses (C3–C5) and ketoses (C5–C7) as acceptors and donors respectively. To improve TKgst activity toward non-phosphorylated aldoses we followed an in vitro evolution strategy based on the active site modification at targeted positions. The three-dimensional structures of microbial TKs such as TKsce,30 TKeco31 and TK from Bacillus anthracis (TKban)32 showed a strong protein sequence homology as evident from superimposition of TKsce, TKeco and TKban active sites.14 The key residues stabilizing the TK substrates are identical and have similar orientation to the cofactor ThDP. Since the three-dimensional structure of TKgst is yet unknown, a homology model of the TKgst active-site pocket containing its natural phosphorylated acceptor aldose, D-erythrose-4phosphate (E4P) was built based on the X-ray crystal structure of TKban as a template, which stems from the same microbial species and has 74% identity (Figure 1).14 With the aim of improving TKgst activity toward (2S)-tetroses such as (2S,3R)-D-threose and (2S,3S)-Lerythrose, having a C2 configuration opposite to that of the natural (2R) acceptor substrate (D-E4P), our strategy was based on our recent results showing that the double site variant L382D/D470S led to 5 fold increased TKgst activity toward the (2S)-triose, Lglyceraldehyde.17 To strengthen this effect, a second generation of TKgst variants was investigated involving Phe435, which is another non-polar residue close to Leu382. To identify plausible positions for the improvement of TKgst toward longer carbon chain aldoses (C5–C6)

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with (2R)-configuration, we took into account the results reported in the literature with other microbial TK sources, TKsce and TKeco, showing that the double variant TKsce R528Q/S527T leads to 2.6-fold and 2.1fold higher activities toward D-ribose and D-glucose, respectively, as compared to wt-TKsce.33 In addition, TKeco variant R520Y was 2.1 times more active than wild-type TKeco against L-arabinose, a diastereoisomer of D-ribose.12d Based on this cumulative evidence and our analysis of the TKgst active site, for further improvement of TKgst activity toward nonphosphorylated C4–C6 aldoses we focused our in vitro evolution strategy on three positions: R521 (R520 in TKeco, R528 in TKsce), S385 (S385 in TKeco, S386 in TKsce) and H462 (H461 in TKeco, H469 in TKsce) .

Figure 1. Model of wild-type TKgst based on the X-ray crystal structure of TKban (PDB entry 3M49) with natural acceptor substrate D-erythrose-4-phosphate (E4P). The model was built using Modeler 9.14 and Chimera.

Site saturation mutagenesis (SSM) was applied for a combined L382X/F435X replacement to improve TKgst activities toward (2S)-tetroses 1 and 2, and a combined R521/S385/H462 replacement was chosen to improve TKgst toward pentoses 3-4 and hexoses 5-6 of (2R)configuration using NDT codon degeneracy (N: cytosine/adenine/guanine/thymine, D: adenine/guanine/thymine; T: thymine). The NDT strategy, which was proposed for the design of “smarter” focused libraries that speed up laboratory evolution by reducing the screening effort, has been

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applied to different enzymes but never to TK.34 The protocol involves 12 codons in comparison to 20 with common NNK usage and reduces the number of amino acids to twelve (Phe, Leu, Ile, Val, Tyr, His, Asn, Asp, Cys, Arg, Ser, Gly), which comprises a balanced mix of polar and nonpolar, aliphatic and aromatic, and negatively and positively charged representatives, while excluding structurally similar amino acids. For an SSM targeted at three positions, the statistical oversampling for 95% coverage of relevant protein sequence space requires screening effort of almost 100,000 clones for complete saturation NNK libraries, as compared to only about 5000 clones with the restricted NDT codon set.35 Characterization of best TKgst variants toward aldoses 1-5. Both TKgst libraries (L382X/F435X and R521X/S385X/H462X) were screened for activity using HPA as donor substrate. (2S)-configured aldoses such as L-glyceraldehyde (C3) and D-threose 1 (C4) were tested with the L382X/F435X library, while higher carbon (2R)-aldoses such as D-ribose 3 (C5), and Dglucose 6 (C6) were tried as possible acceptor substrates of the R521X/S385X/H462X library. We used an efficient, direct, quantitative highthroughput colorimetric assay developed earlier, which is based on a pH shift caused upon TK catalyzed HPA consumption in the presence of phenol red as pH indicator.35 The decarboxylation of HPA, the donor substrate, causes release of bicarbonate and the pH to rise, rendering measurements independent of the structure of the acceptor substrate. Absorbance variation at 560 nm was measured at initial time and after 30 minutes of reaction at room temperature (phenol red was found unstable at 60°C). The TKgst variants leading to the best activities toward the selected acceptor substrates were sequenced and purified by Ni2+ chelating affinity column chromatography and characterized by their specific activities with aldoses 1-5 and HPA as donor substrate in comparison with wild-type TKgst (Chart 1). Taking into account the concentrations of aldoses (0.02 M–0.05 M for C3 and C4, respectively and 0.2 M for C5 and C6), we noted that activity of wild-type TKgst decreased expectedly with increasing carbon chain length.

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A)

Specific activity (U.mg-1)

0.6 0.5 0,41

0.4 0.3 0,17

0.2

0,11

0,10

0.1

0,05

0,03

0 L-glyceraldehyde

C3

B) 1.4 Specfic activity (U.mg-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

D-threose

1 C4

L-erythrose

2 C4

1,21

1.2 1 0.8 0,51

0.6 0.4

0,34

0,33 0,24

0.2

0,15

0,10 0.0010,024

0 D-ribose

3 C5

D-xylose

4 C5

D-glucose

6 C6

Chart 1. Specific activities A) of wild-type ■ and L382F/F435Y ■ with L-glyceraldehyde C3, D-threose 1, Lerythrose, 2 and B) of wild-type ■ R521Y/S385/H462N ■ and R521V/S385D/H462S ■ with D-ribose 3, D-xylose 4, D-glucose 5.a) The assay solution contained 0.02 M of L-glyceraldehyde, 0.05 M of D-threose, 0.05 M of L-erythrose, 0.2 M of D-ribose, 0.2 M of D-xylose and 0.2 M of D-glucose, 20 – 90 mg of wta)

TKgst and TKgst variants, 1 mM of ThDP, 1 mM of MgCl2, 84 µM of phenol red and 2 mM of triethanolamine buffer at pH 7.15 and at 25°C. The double variant L382F/F435Y was found to be 4 times more active than wild-type TKgst toward (2S)glyceraldehyde, which was used as a model substrate for the screening of the L382X/F435X library. This variant was further tested toward both (2S)-tetroses, Dthreose 1 and the (3S)-epimer L-erythrose 2, and led to a 3.6 and 3.7 fold increase in activity, respectively, compared with wild-type TKgst (Chart 1A). We note

that the C3 configuration of 1-(3R) and 2-(3S) only had a minor influence on the activity of both the wild-type TKgst and its L382F/F435Y variant. The double TKgst variant R521Y/S385/H462N showed the greatest improvement toward D-ribose 3 and its (3S)-epimer D-xylose 4, with a 3.5 fold and 3.3 fold increase in activity, respectively, compared with wild-type TKgst (Chart 1B). This TKgst variant was more efficient toward D-ribose 3 than TKsce R528Q/S527T previously reported in the literature, which only lead to a 2.6 fold increase in activity compared with wildtype TKsce.34 We note that the presence of a Tyr residue in place of Arg at position 521 positively influences the activity of TKgst, which parallels a previous observation for an enhanced activity of TKeco R521Y variant toward L-arabinose, another pentose.12d The influence of the absolute configuration of C3 on the activity of wild type TKgst and both R521Y/S385/H462N and R521V/S385D/H462S variants is more pronounced with pentoses than with tetroses as indicated by the higher activities with (3R)-D-ribose 3 in comparison with its (3S)-epimer, D-xylose 4 (Chart 1B). Finally, the triple variant TKgst R521V/S385D/H462S displays useful activity also toward D-glucose 6, even though the specific activity is low (0.15 U·mg-1), while this activity is undetectable with wild-type TKgst (Chart 1B). To explain these experimental results, we used molecular modeling to analyze the interactions between the best identified substrates from each series (i.e., D-threose 1 C4, D-ribose 3 C5 and D-glucose 6 C6) and the active site residues of the three best TKgst variants L382F/F435Y, R521Y/S385/H462N and R521V/S385D/H462S, respectively. Three models (SI) were constructed for binding of the aldoses in a fashion similar to the model previously constructed for binding of the natural substrate E4P by wild-type TKgst (Figure 1). When studying interactions of D-threose 1 in the active site of L382F/F435Y (SI), we noted that an (S)configured hydroxyl group at C2 of 1 could form a new hydrogen bond with the phenolic hydroxyl group upon the polar replacement Phe435Tyr. In addition, a Phe residue in place of Leu 382 could benefit from additional stacking interactions with Tyr435, improving the positioning of the latter. In the case of TKgst variant R521Y/S385/H462N (SI), the hydroxyl group on C5 of D-ribose 3 could interact

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with the hydroxyl group of Tyr at position 521 and the oxygen of the Asn residue at position 462 by hydrogen bonding via a crystallographic water molecule. A third direct hydrogen bond is possible between Ser 385 and the hydroxyl group at C4 of D-ribose 3. This strong network of hydrogen bonds might be more favorable for substrate binding than that observed in the active site of wild-type TKgst, which would explain the experimental increase in activity with TKgst variant R521Y/S385/H462N by an increased substrate affinity (Chart 1B). In the case of the triple mutant R521V/S385D/H462S (SI), the Val replacement at position 521 could significantly enlarge the entrance to the active site pocket, improving the accessibility for an aldose having 6 carbon atoms. The hydroxyl group on C6 of D-glucose 6 could be stabilized by hydrogen bonding with Glu 469, itself stabilized by Ser 26. In addition, a direct hydrogen bond seems to be more favored between the hydroxyl function on C5 of Dglucose 6 and Asp 385 than with the native Ser in the wild-type TKgst active site. In conclusion, TKgst variant R521V/S385D/H462S creates a hydrogen bond network in the active site that is appropriate to stabilize an openchain D-glucose 6 molecule in an extended conformation, which could therefore explain its ability to accept this compound as a free aldehyde substrate, whereas this aldose is not converted by wild-type TKgst (Chart 1B). Synthesis of ketoses 7-12 with TKgst variants. To validate the analytical results, preparative-scale syntheses were performed with HPA as donor and the selected aldoses 1-6 (C4–C6) as acceptors (Tables 1 and 2). All reactions were carried out at 60°C, in the presence of the most appropriate TKgst variant, such as L382F/F435Y with aldoses 1 and 2, R521Y/S385/H462N with 3 and 4 and R521V/S385D/H462S with 5 and 6. A study of the thermostability of TKgst variants showed a total recovery of enzymatic activity after 5 days at 60°C. At this temperature, HPA showed acceptable stability (80% of remaining HPA after 24 h at 60°C), while TKgst activity was 7 times higher than at 20°C, thus allowing significantly shorter reaction times.14 Aldoses 1-6 were stable in aqueous solution at 60°C (data not shown). We note that in the cyclization equilibrium the fraction of the linear aldose form having a free aldehyde group is rising with increasing

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temperatures for entropic reasons, and thus becomes better accessible for the enzymatic reaction.37 The pH was kept constant by pH-stat control. The consumption of aldoses 1-6 was quantified by 1H NMR analysis of aliquots taken from the reaction mixture at certain time intervals, allowing the measurement of the final conversion levels of aldoses (Table 2). The conversion of tetroses 1 and 2 by L382F/F435Y and pentoses 3 and 4 by R521Y/S385/H462N went to completion in 48 h, 72 h, 32 h and 96 h, respectively. In all cases the corresponding products 7-10 were formed exclusively and isolated with yields of 62%, 84%, 67% and 59%, respectively. The high diastereoselectivity (no other stereoisomer detectable; d.e. > 95%) was verified by 13C NMR analysis and optical rotation data, which were identical to those obtained with wild-type TKgst and reported in the literature. These results indicate that TKgst variants consistently led to the formation of 7, 8 L-erythro-(3S, 4S)-ketoses and 9, 10 Dthreo-(3S, 4R)-ketoses. We note that the unprotected product 10 has not been described before. The somewhat lower efficiency of the reaction with aldose substrates 2 and 4 correlates with longer reaction times required to reach complete conversion (72h and 96 h respectively) seems to be related with the C3-(S)configuration of 2 and 4, which is less favorable for the activity than the C3-(R)-configuration present in 1 and 3. This result is in accordance with the data obtained at analytical scale, particularly for variant R521Y/S385/H462N showing 4-fold lower activity with 4 compared to 3. The conversion of hexoses 5 and 6, impossible with wild-type TKgst even by incubation for several days at 60°C, was achieved at this temperature by using the variant R521V/S385D/H462S, although this required significantly longer reaction times than for tetroses 1, 2 and pentoses 3, 4. The conversion of 5 was almost complete in 7 days, while only 76% of 6 was transformed in 21 days. As stated before, the C3-(R)configuration in 5 appeared more favorable compared with its C3-(S) epimer 6. In addition, the very high stability of the all-equatorial substitution pattern in the pyranose form of glucose 6 renders its aldehyde form, the real enzyme substrate, difficult to access. Unprotected pure ketose 11, never described in the literature before, was obtained with an isolated yield of 70% with no indication for the presence of another

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diastereoisomer. Ketose 12 was identified in the reaction mixture by in situ 1H NMR (SI). Table 2. Preparative-scale synthesis of ketoses 7-12 from aldoses 1-6 catalyzed by engineered TKgst variants. O HO

OH CO2H

+

R O

HPA Donor

Cn Aldose acceptora 1-C4

Cn+2 Ketose producta 7-C6

2-C4

OH

HO ThDP, Mg2+

C4-6 aldoses Acceptor

TKgst variantsa

O

TKgst variant

R

+

CO2

OH C6-8 ketoses Product

Reaction time

L382F/F435Y

Aldose conversion (%)b > 95

d.e. (%)d

48h

Isolated yield (%) 62

8-C6

L382F/F435Y

> 95

72h

84

> 95

3-C5

9-C7

R521Y/S385/H462N

>95

32h

67

> 95

4-C5

10-C7

R521Y/S385/H462N

>95

96h

59

> 95

5-C6

11-C8

R521V/S385D/H462S

93

7 days

70

> 95

6-C6

12-C8

R521V/S385D/H462S

76

21 days

ndc

ndc

> 95

aReactions

were carried out with 6-30 mg of TKgst variants, 0.1 mM of ThDP, 1 mM of MgCl2, 50 mM of aldose, 50-550 mM of LiHPA at pH 7 and at 60°C. b Aldose acceptor conversion determined by in situ IH NMR analysis. cNot determined, in situ 1H NMR of reaction mixture (SI). d Diastereoisomeric excess (d.e.) determined by 1H NMR.

Furthermore, all reactions catalyzed by TKgst variants L382F/F435Y andR521Y/S385/H462N were compared to the corresponding conversions catalyzed by wild-type TKgst performed with equimolar concentrations of donor and acceptor substrates 1-4 (SI). Consistently, higher conversion rates of aldoses 14 and higher isolated yields of ketose products 7-10 were obtained with TKgst variants than with the wildtype enzyme. The formation of 11 and 12 from 5 and 6, respectively, was not observed with wild-type TKgst but only with the variant R521V/S385D/H46S.

CONCLUSION In summary, we have developed an efficient, stereoselective, two-carbon elongation procedure catalyzed by engineered TKgst variants for the conversion of six non-phosphorylated aldoses 1-6 with various carbon backbone lengths (C4-C6). To enhance the TKgst activity, two variant libraries (L382X/F435X and R521/S385/H462) were built by SSM using NDT

codon degeneracy on targeted positions that were determined with the aid of a homology model of the wild-type TKgst active site. Three best TKgst variants showed improved activities compared with wild-type TKgst toward tetroses 1-2 (L382F/F435Y), pentoses 3-4 (R521Y/S385/H462N) and hexoses 5 and 6 (R521V/S385D/H462S). These TKgst variants were applied in a one-pot, one-step procedure performed at 60°C under near-neutral pH, which allowed a twocarbon elongation of aldoses 1-6 (C4-C6) with HPA as ketol donor leading to the corresponding naturally rare ketoses 7-12 (C6–C8) that have important biological functions. The ketoses 7-11 were synthesized with excellent diastereoselectivity in reasonable reaction times. Total conversion of aldoses 1-4 was observed and almost total conversion of both hexoses 5-6. We note that free ketoses 10, 11 and 12 with unmodified hydroxyl groups were not hitherto described in the literature except for phosphorylated or protected derivatives.

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This procedure is an attractive alternative to chemical strategies and is superior to other enzymatic approaches that utilize phosphorylated sugars as substrates and intermediates, and that require additional enzymes to liberate free sugars. These results set the path to enhance TKgst activities toward other aldose stereoisomers for obtaining corresponding high-carbon ketoses.

EXPERIMENTAL SECTION General data. Chemicals and solvents were purchased from Sigma-Aldrich and CarboSynth. L-glyceraldehyde was obtained from Molekula Society. Li-HPA was synthesized according to the procedure described in the literature.36 Bradford reagent was procured by BioRad. Proteins and enzymes were acquired from SigmaAldrich. BugBuster solution, lysozyme and Benzonase endonuclease were provided by Merck, Germany. The 96-wells microplates and deep well 96-wells plates were shaken and incubated in a Titramax 1000 incubator from Heidolph. Lyophilisation was carried out with Triad LABCONCO dryer. UV-visible absorbances were measured using a Spark control 10 plate reader from TECAN and a spectrophotometer Agilent Technologies, Cary 300 UV-Vis. MARCHEREYNAGEL GmbH & Co KG 60 F254 silica gel TLC plates and MARCHEREY-NAGEL GmbH & Co KG 60/40-63 mesh silica gel for Liquid Flash Chromatography were used. NMR spectra were recorded in D2O or in CD3OD on a 400 MHz Bruker Avance III HD spectrometer or a 500 MHz Bruker AC-500 spectrometer. Chemical shifts were referenced to the residual solvent peak. The following multiplicity abbreviations were used: (s) singlet, (d) doublet, (t) triplet, (m) multiplet. The optical rotation was determined with a polarimeter P2000 JASCO PTC-262 at the given temperature and wavelength (Na-D-line 589 nm) in a cell 10 cm long cell; [α] values are given in 10-1 deg cm2 g-1 (concentration given as g.100 mL-1). Oligonucleotides were synthesized by Eurofins Genomics (Ebersberg, Germany). QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, USA) was used to build the TKgst mutants libraries. Gene sequencing was performed by Eurofins Genomics (Ebersberg, Germany). Plasmidic DNA was extracted with the GenElute™ Plasmid Miniprep kit or with the GenElute™ Plasmid Midiprep kit (Sigma-Aldrich).

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Modeling of TKgst structure. Model of wild-type TKgst active site pocket was built by sequence homology from the X-ray three-dimensional structure 3M49 of TK from Bacillus anthracis (TKban) as template using Modeller9v14 software and chimera with the optional inclusion of water molecules. The resulting model was geometrically optimized by energy minimization using sybylx2.1(Tripos International, St. Louis, MO, USA) software (forcefield=Tripos, method= conjugated gradient, gradient=0.05, charge=Gasteiger-Huckel, dielectric constant=4.0). Models Leu382PhePhe435Tyr, Arg521Tyr-His462Asn and Arg521VaLSer385Asp-His462Ser TKgst active site pockets were obtained using rotamer module of Chimera, and optimized by sybylx2.1 under the same conditions as mentioned previously. Construction of TKgst variant libraries. For the double site L382X/F435X and triple site R521X/S385X/H462X libraries appropriated oligonucleotides were designed (SI). For the PCR, the artificial TKgst gene was used in the vector pET47b (pET47b-TKgst) as template. The PCR products were transformed by thermic shock into XL10 Gold competent cells and cultured in NZY+ medium with 30 μg.mL-1 of kanamycin. The plasmids were extracted for sequencing to evaluate the quality of the TKgst mutants libraries. The plasmids were then transformed by thermic shock into competent BL21(DE3) cells. The resultant clones were cultured on LB-kanamycin agar plates overnight. A total of 465 and 5022 colonies for double and triple site libraries, respectively, were picked into 96-well plates. The whole plates were sealed with plastic lids and stored at - 80°C. Screening method of TKgstvariant libraries The cell pellets were resuspended in lysis buffer (150 µL) and incubated at 37°C, 900 rpm for 1 to 2 hours (SI). The cell lysates were centrifuged at 4.000 rpm for 1 hour. Then, aliquots of the supernatant were transferred to a new 96-well plate. A solution (100L) containing 1 mM of MgCl2, 1 mM of ThDP, 84 µM of phenol red, in 2 mM triethanolamine buffer at pH 7.15 was added to each well. Aldehyde solution (20 mM of L-glyceraldehyde, 200 mM of D-ribose, 200 mM of D-xylose and 200 mM of D-glucose as final concentrations) was then introduced to each well. The reaction was initiated by the addition

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ACS Catalysis

of Li-HPA (10mM, 10 L). A multichannel pipetting workstation TECAN EVO 150 was used for automation of the process. The absorbance was read by platereader at 560 nm immediately and after 30 minutes of reaction at 25°C.35 Expression and purification of positive TKgst variants TKgst variants. A 100 μL culture aliquot of each of the clones was transferred into 100 mL LB medium containing 30 µg.mL-1 of kanamycin and cultured at 37°C, 250 rpm, overnight. Then, the cells were harvested by centrifugation (8.000 rpm, 15 minutes, 4°C), washed three times with 50 mM phosphate buffer containing 300 mM of NaCl (pH 8.0), and finally harvested (4.000 rpm, 4°C, 15 minutes). Cells were stored at –80°C. A total of 1.23 to 1.58 g of cells (wet weight) was obtained from each culture (2.25 g. L-1 of culture). The 3 His6-tagged TKgst variants L382F/F435Y, R521Y/S385/H462N and R521V/S385D/H462S were purified by Ni2+ chelating affinity (SI). The protein containing fractions were collected and dialysed against 2 mM of triethanolamine buffer over-night at pH 7.5 and then against water during 5 hours at pH 7.5 through dialysis tubing. After purification from 600 mL of cell culture, a total quantity of 94 to 115 mg of protein was obtained from 1.23 to 1.58 g of cells, respectively, with a total activity of TKgst variants of 0.4 – 142 U, corresponding to a specific activity of 0.006 – 2 U mg−1, at 25 °C (SI). Proteins were then lyophilized from aqueous solution at pH 7.5 and stored at 4°C. The sample purity was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SI). Wild-type TKgst and TKgst variants activity. One unit of TKgst activity is defined as the amount of TK that catalyzes the formation of 1 μmol of ketose product per minute at 25 °C in 0.1 M glycylglycine buffer at pH 7.5. TKgst enzymatic assay was performed in the presence of L-erythrulose and D-ribose-5-phosphate (D-R5P) leading to D-sedoheptulose-7-phosphate (D-S7P) and glycolaldehyde (GA).37 The GA formed is reduced by yeast alcohol dehydrogenase (ADH, EC 1.1.1.1) to ethylene glycol in the presence of nicotine adenine dinucleotide, reduced form (NADH). The disappearance of NADH was followed by spectrophotometry at 340 nm (value of εNADH at 340 nm is 6220 M−1. cm−1).

TKgst variants thermostability profiles. The thermostability of TKgst variants L382F/F435Y, R521Y/S385/H462N and R521V/S385D/H462S was studied at 60°C with L-erythrulose and D-R5P as substrates. Enzyme solutions were incubated in 0.1 M glycylglycine buffer at pH 7,5 and at 60°C, in 1.5 mL tubes for up to 96 hours in an incubator and stirred at 150 rpm. The residual activities were measured during 5 days by the standard assay described above.40 In situ 1H NMR measurements. Progress of preparative scale enzymatic synthesis was monitored by using quantitative in situ 1H NMR spectroscopy relative to 3-trimethylsilyL-2,2,3,3tetradeuteropropionate (TSP-d4) as internal standard for δ (ppm) = 0.0. 450 µL aliquots of reaction medium were removed at defined intervals and mixed with 50 µL of TSP-d4 (50 mM, 8.5 mg.mL-1 in D2O). Preparative scale enzymatic synthesis of ketoses. General procedure. 0.1 mM of ThDP and 1 mM of MgCl2·6H2O were dissolved in H2O and the pH was adjusted to 7 with 0.1 M of NaOH. To this stirred solution was added wt-TKgst or TKgst variant (6 mg) and the mixture was stirred for 20 minutes at 60°C. In another flask, 50 mM of Li-HPA and 50 mM of acceptor substrate were mixed in H2O and adjusted to pH 7 with 0.1 M NaOH. After preincubation of enzyme and cofactors, Li-HPA and acceptor substrate were added, and the mixture was stirred at 60°C. The final volume was 20 mL. The pH was maintained at 7 by adding 0.1 M HCl using a pH stat (Titroline® 7000, SI Analytics). The reaction was monitored by measuring HPA and acceptor substrate consumption by in situ 1H NMR and TLC. After total conversion of HPA (12 h-21 days), silica was added and the reaction mixture was concentrated to dryness under reduced pressure, then loaded onto a silica column. After silica gel chromatography using CH2Cl2/CH3OH, 90:10 v:v as eluent (for D-altro-heptulose 9, D-ido-heptulose 10 and D-glycero-D-altro-otulose 11) and ethyl acetate:CH3OH, 100:0-90:10 v:v as eluents (for D-tagatose 7 and Lpsicose 8), ketose prducts 7-11 were isolated and characterized. (3S,4S,5R)-1,3,4,5,6-Pentahydroxyhexan-2-one (Dtagatose) 7. Compound 7 was isolated as a white powder (23 mg, yield: 13 % with wt-TKgst; 77 mg, yield: 43 %, with TKgst variant L382F/F435Y; 111 mg, yield: 62

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%, with TKgst variant L382F/F435Y). TLC: Rf 0.31 (CH2Cl2:CH3OH, 80:20 v:v). [α]H2O = –2.3 (c 0.1 H2O) with wt-TKgst; [α]D20 = –4.0 (c 0.3 H2O) with TKgst variant L382F/F435Y; lit: [α]D20 = –2.9 (c 0.9, CH3OH).38 NMR data for 3 obtained with either TKgst were identical to those previously described39 (ratio: α-Dtagato-2.6-pyranose/β-D-tagato-2.6-pyranose: 80/20; lit. ratio: α-D-tagato-2.6-pyranose/β-D-tagato-2.6pyranose/α-D-tagato-2.5-furanose/β-D-tagato-2.6furanose: 79/14/2/5.42 1H NMR (400 MHz, D2O): α-Dtagato-2,6-pyranose = 3.86 (d, J=2.7Hz, 1H, H3), 3.843.79 (m, 2H, H5, H4), 3.72 (d, J=10.9Hz, 1H, H6a), 3.69 (d, J=11.8Hz, 1H, H1a), 3.60 (d, J=10.8Hz, 1H, H6b), 3.47 (d, J=11.8Hz; 1H, H1b); β-D-tagato-2,6-pyranose δ = 4.15 (dd, J=13.1Hz, J=1.8Hz, 1H, H6a), 3.99 (t, J=3.4Hz, 1H, H4), 3.91 (d, J=3.5Hz, 1H, H3), 3.89 (dt, J=4.1Hz, J=2.0Hz ,1H, H5), 3.66 (d, J=11.8Hz, 1H, H1a), 3.58-3.56 (m, 1H, H6b), 3.46 (d, J=11.8Hz; 1H, H1b). 13C NMR (101 MHz, D2O): α-D-tagato-2,6-pyranose δ = 99.0 (C-2), 71.7 (C-4), 70.6 (C-3), 67.2 (C-5), 64.7 (C-1), 63.0 (C-6) ; β-D-tagato-2,6-pyranose δ = 99.1 (C-2), 70.6 (C-4), 70.1 (C-5), 64.5 (C-3), 64.3 (C-1), 60.9 (C-6). m/z HRMS found [M + Cl]- 215.0322, C6H12O6Cl requires 215.0317 (obtained with wt-TKgst); m/z HRMS found [M + Cl]215.0322, C6H12O6Cl requires 215.0317 (obtained with TKgst variant L382F/F435Y). (3S,4S,5S)-1,3,4,5,6-Pentahydroxyhexan-2-one (Lpsicose) 8. Compound 8 was isolated as a white powder (18 mg, yield: 10 % with wt-TKgst; 99 mg, yield: 55 % with TKgst variant L382F/F435Y; 151 mg, yield: 84 %, with TKgst variant L382F/F435Y). TLC: Rf 0.31 (CH2Cl2:CH3OH, 80:20 v:v). [α]D25 = –2.3 (c 0.1 H2O) with wt-TKgst ; [α]D20 = –1.4 (c 2 H2O) with TKgst variant L382F/F435Y; lit. [α]D20 = –2.4.40 1H NMR data for 8 obtained with either TKgst were identical to those previously described for the D-enantiomer.41,42 Ratio: αL-psico-2.6-pyranose/β-L-psico-2.6-pyranose/α-L-psico2.5-furanose/β-L-psico-2.6-furanose: 26/22/38/14; lit. ratio for corresponding D-enantiomer: 26/21/38/15.42 1H NMR (400 MHz, D2O) : α-L-psico-2,6-pyranose δ = 4.18 (dd, J=3.5Hz, J=1.7Hz, 1H, H4), 3.83-3.79 (m, 1H, H6a), 3.82 (d, J=1.1Hz, 1H, H5), 3.68 (d, J=1.9Hz, 1H, H3), 3.66 (d, J=6.5Hz, 1H, H1a), 3.65-3.62 (m, 1H, H6b); 3.41 (d, J=11.7Hz, 1H, H1b); β-L-psico-2,6-pyranose δ = 4.07-4.02 (m, 2H, H6), 3.99 (t, J=3.3Hz, 1H, H4), 3.93 (dd, J=3.3Hz, J=1.7Hz, 1H, H5), 3.80-3.76 (m, 1H, H1a), 3.78 (d, J=1.4Hz, 1H, H3), 3,58-3.52 (m, 1H, H1b); α-L-psico-

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2,5-furanose δ = 4.11-4.06 (m, 3H, H5, H4, H3), 3.77-3.71 (m, 1H, H6a), 3.63-3.58 (m, 1H, H6b), 3.58-3.53 (m, 1H, H1a/b); β-L-psico-2,5-furanose δ = 4.32 (dd, J=7.7Hz, J=4.7Hz, 1H, H4), 4.11-4.05 (m, 1H, H5), 4.01 (d, J=4.7Hz, 1H, H3), 3.83-3.79 (m, 2H, H1), 3.69-3.61 (m, 2H, H6). 13C NMR (101 MHz, D2O) : α- L-psico-2,6pyranose δ = 98.6 (C-2), 72.6 (C-4), 66.7 (C-5), 66.3 (C3), 64.0 (C-1), 58.8 (C-6); β-L-psico-2,6-pyranose δ = 99.3 (C-2), 71.1 (C-3), 69.9 (C-5), 65.9 (C-4), 65.1 (C-6), 64.8 (C-1); α-L-psico-2,5-furanose δ = 104.1 (C-2), 83.6 (C-5), 71.2 (C-4), 71.2 (C-3), 64.1 (C-1), 62.2 (C-6) ; β-Lpsico-2,5-furanose δ = 106.5 (C-2), 83.6 (C-5), 75.5 (C3), 71.9 (C-4), 63.7 (C-6), 63.2 (C-1). m/z HRMS found [M + Cl]- 215.0322, C6H12O6Cl requires 215.0317 (obtained with wt-TKgst); m/z HRMS found [M + Cl]215.0322, C6H12O6Cl requires 215.0317 (obtained with TKgst variant L382F/F435Y). (3S,4R,5R,6R)-1,3,4,5,6,7-Hexahydroxy-heptan-2one (D-altro-heptulose) 9. Compound 9 was isolated as a colorless oil (92 mg, yield: 44 % with wt-TKgst; 141 mg, yield: 67 % with TKgst variant R521Y/H462N). TLC: Rf 0.22 (CH2Cl2: CH3OH, 80:20 v:v). [α]D20 = + 7.8 (c 0.7 H2O) with wt-TKgst ; [α]D20 = + 5.9 (c 0.7 H2O) with TKgst variant R521Y/H462N ; lit. [α]D20 = + 8.6 (c 0.1, H2O).15 NMR data for 9 obtained with either TKgst were identical to those previously described.43 Ratio: β-Daltro-heptulo-2,5-furanose/α-D-altro-heptulo-2,6pyranose/α-D-altro-heptulo-2,5-furanose = 67/19/14; lit. ratio = 64/20/16.19 1H NMR (400 MHz, D2O) : β- D-altroheptulo-2,5-furanose δ = 4.30-4.23 (m, 1H, H4), 4.05 (d, J= 7.9 Hz, 1H, H3), 3.80 (d, J=3.3 Hz, 1H, H6), 3.73-3.72 (m, 1H, H5), 3.63-3.57 (m, 2H, H7a/b), 3.53 (d, J= 12.1 Hz, 1H, H1a), 3.54 (d, J=12.1 Hz; H1b); α-D-altroheptulo-2,6-pyranose δ = 4.04 (d, J=7.4 Hz, 1H, H4), 3.98 (m, 1H, H6), 3.91 (d, J=3.7 Hz, 1H, H3), 3.83 (dd, J=4.1 Hz, J=1.9 Hz, 2H, H7a/b), 3.81 (m, 1H, H5), 3.65 (d, J=11.7 Hz, 1H, H1a), 3.46 (d, J= 11.7 Hz, 1H, H1b); α-Daltro-heptulo-2,5-furanose δ = 4.14 (dd, J=6.1 Hz, J=4.5 Hz, 1H, H4), 3.94 (d, J=2.6 Hz, 1H, H3), 3.88-3.84 (m, 2H, H5, H6a), 3.79-3.75 (m, 1H, H6b), 3.69 (d, J=9.3 Hz, 1H, H1a), 3.51 (d, J= 11.7 Hz, 1H, H1b). 13C NMR (101 MHz, D2O) : β-D-altro-heptulo-2,5-furanose δ = 101.6 (C-2), 80.1 (C-5), 75.6 (C-3), 75,3 (C-4), 72.6 (C-6), 62.4 (C-1), 62.2 (C-7); α-D-altro-heptulo-2,6-pyranose δ = 97.4 (C-2), 70.8 (C-4), 68.7 (C-6), 68.2 (C-3), 64.0 (C-1), 63.4 (C-5), 61.2 (C-7); α-D-altro-heptulo-2,5-furanose δ = 104.6 (C-2), 81.7 (C-3), 81.6 (C-5), 76.4 (C-4), 71.5 (C-

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ACS Catalysis

6), 62.7 (C-1), 62.3 (C-7). m/z HRMS found [M + Cl]245.0431, C7H14O7Cl requires 245.0656 (obtained with TKgst variant R521Y/H462N). (3S,4R,5S,6R)-1,3,4,5,6,7-Hexahydroxy-heptan-2one (D-ido-heptulose) 10. Compound 10 was isolated as a colorless oil (44 mg, yield: 21 % with wt-TKgst; 77 mg, yield: 37 %, with TKgst variant R521Y/H462N; 124 mg, yield: 59 %, with TKgst variant R521Y/H462N). TLC: Rf 0.22 (CH2Cl2:CH3OH, 80:20 v:v). [α]D20 = –36.4 (c 0.3 H2O) with wt-TKgst ; [α]D20 = –35.8 (c 0.4 H2O) with TKgst variant R521Y/H462N ; lit: [α]D20 = –34 ± 8 (c 0.3, H2O).44 NMR spectra showed the presence of five isomeric forms : two five-membered (furanose) and two six-membered (pyranose) species, corresponding to the α and β anomers and one acyclic species. The ratio was determined but α and β anomers were not attributed for each cyclic species. Ratio: 60 (furanose)/16 (pyranose)/12 (furanose)/8 (pyranose)/4 (acyclic). 1H NMR (400 MHz, D2O): major cyclic form δ = 4.39-4.34 (m, 1H, H4), 4.19 (dd, J= 5.9 Hz, 4.8 Hz, 1H, H5), 4.15 (d, J=5.6 Hz, 1H, H3), 3.93-3.87 (m, 1H, H6), 3.67 (d, J=4.4 Hz, 2H, H1a/b), 3.63 (d, J=6.8 Hz, 2H, H7a/b). 13C NMR (101 MHz, D2O) : major cyclic form δ = 101.8 (C2), 77.1 (C5), 76.2 (C3), 75.4 (C4), 70.0 (C6), 63.3 (C7), 62.6 (C1). m/z HRMS found [M + Cl]245.0429, C7H14O7Cl requires 245.0656 (obtained with wt-TKgst) ; m/z HRMS found [M + Cl]- 245.0430, C7H14O7Cl requires 245.0656 (obtained with TKgst variant R521Y/H462N). (3S,4R,5S,6R,7R)-1,3,4,5,6,7,8-Heptahydroxyoctan-2one (D-glycero-D-altro-octulose) 11. Compound 11 was isolated as a colorless oil (35 mg, yield: 70 % with TKgst variant R521V/S385D/H462S). TLC: Rf 0.15 (CH3COOC2H5:CH3OH, 70:30 v:v). NMR spectra showed the presence of four isomeric forms: two fivemembered (furanose) and two six-membered (pyranose) cyclic species corresponding to the α and β anomers. The ratio was determined but α and β anomers were not attributed for each species: 64 (furanose) a /15 (furanose) b /14 (pyranose) c / 7 (pyranose) d. 1H NMR (500 MHz, D2O): major furanose form a δ = 4.38 (t, J=7.5 Hz, 1H, H4), 4.14 (d, J=7.9 Hz, 1H, H3), 4.0 (dd, J=7.1Hz, 4.9Hz, 1H, H5), 3.89-3.87 (m, 1H, H7), 3.86-3.84 (m, 1H, H6), 3.88-3.82 (dd, 1H, J = 11.8 Hz, 3.0 Hz, 1H, H8a), 3.73-3.68 (dd, J=11.8 Hz,6.4 Hz, 1H, H8b), 3.64 (d, J=12.1 Hz, 1H H1a/b), 3.59 (d, J=12.1Hz, 1H, H1a/b); furanose form b δ = 4.29-4.24 (m, 2H, H4 and H5), 4.14 (d, J=4.5 Hz, 1H, H3), 3.91-3.87 (m, 1H, H7), 3.77-3.73 (m, 1H, H6), 3.70 (d, J=11.7, 1H,

H1a) 3.53 (d, J=11.7 Hz, 1H, H1b), pyranose form c δ = 4.08 (dd, J = 7.3Hz,3.4 Hz, 1H, H4), 3.98 (dd, J =10.4 Hz, 3.4Hz, 1H, H5), 3.97 (d, J = 3.4 Hz,1H, H3); minor pyranose form d δ = 4.35 (dd, J = 2.0Hz, 3.3 Hz, 1H, H5), 4.19-4.15 (m, 1H, H7), 4.13-4.10 (dd, J=3.3Hz, 10.0Hz, 1H, H4), 3.91 (d, J=10.0 Hz, 1H, H3), 3.85 (dd, J=2.0Hz, 10Hz, 1H, H6), 3.74 (d, J=11.6 Hz, 1H, H1a), 3.58 (d, J=11.6 Hz, 1H, H1b), 3.73-3.68 (m, 2H, H8). 13C NMR (101 MHz, D2O) : major furanose form a δ = 101.6 (C2), 80.3 (C5), 75.8 (C3), 74.9 (C4), 72.0 (C7), 71.9 (C6), 62.7 (C8), 62.6 (C1); furanose form b δ = 104.7 (C2), 81.9 (C3), 81.7 (C4 or C5), 76.0 (C4 or C5), 70.9 (C6), 67.1 (C7), 62.9 (C1 or C8), 62.7 (C1 or C8); pyranose form c δ = 97.5 (C2), 72.4-71.9 (C6 or C7), 71.2 (C4), 68.5 (C3), 64.3 (C4), 64.2 (C1 or C8), 61.9 (C1 or C8); minor pyranose form d δ = 99.2 (C2), 78.7 (C6), 70.7 (C7), 68.7 (C5), 67.1 (C4), 67.0( C3), 64.0 (C1), 63.1 (C8). m/z HRMS found [M + Cl]- 245.0546, C8H16O8Cl requires 275.0539 (obtained with TKgst variant R521V/S385D/H462S).

ASSOCIATED CONTENT Supporting Information.

This information is available free of charge on the ACS Publications website. Data of TKgst variant production, data of TKgst-catalyzed reactions, active site models of TKgst variants, 1H NMR and 13C NMR spectra of ketose products 7-12.

AUTHOR INFORMATION Corresponding Authors *[email protected] *[email protected] ORCID Laurence Hecquet : 0000-0003-2971-5686 Franck Charmantray : 0000-0001-8638-8349 Wolf-Dieter Fessner : 0000-0002-9787-0752

ACKNOWLEDGMENTS This work was funded by the Agence Nationale de la Recherche (grant ANR-09-BLAN-0424-CSD3 to L.H.), the Fonds Régional Innovation Laboratoire (grant DOS00494484/00, to L.H.) and the Deutsche Forschungsgemeinschaft (grant Fe244/10-1 to W.D.F.).

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Manisha, J. P.; Rekha, C. A.; Arti, T. P.; Samir, R. D.; Darshan, H. P. A Single and Two Step Isomerization Process for D-Tagatose and L-Ribose Bioproduction using L-Arabinose Isomerase and D-Lyxose Isomerase. Enzyme Microb. Technol. 2017, 97, 27-33. (23) Leang, K.; Sultana, I.; Takada, G.; Izumori, K.A. A Novel Bioconversion of L-Fructose to L-Glucose by Klebsiella pneumoniae. J. Biosci. Bioeng. 2003, 95, 310-312. (24) (a) Itoh, H.; Izumori, K. Enzymatic Production of LTagatose and L-Fructose from L-Sorbose and L-Psicose, Respectively. J. Ferment. Bioeng. 1996, 81, 351–353. (b) Dibello, E.; Gamenara, D.; Seoane, G.A.; Efficient Synthesis of Orthogonally Protected Rare l-Hexoses and Derivatives. Synthesis 2017, 5, 1087-1092. (25) Wamelink, M. M.; Struys, E. A.; Jansen, E. E. W.; Blom, H.; Vilboux, T.; Gahl, W. A.; Komhoff, M.; Jakobs, C.; ELevtchenko, E. N. Elevated Concentrations of Sedoheptulose in Bloodspots of Patients with Cystinosis Caused by the 57-kb Deletion: Implications for Diagnostics and Neonatal Screening. Mol. Genet. Metab. 2011, 3, 339-342. (26) (a) Wamelink, M. M. C.; Struys, E.A.; Valayannopoulos, V.; Gonzales, M.; Saudubray, J.‐M.; Jakobs, C. Retrospective Detection of Transaldolase Deficiency in Amniotic Fluid: Implications for Prenatal Diagnosis. Prenat. Diagn. 2008, 5, 460-462. (b) Ranoux, A.; Isabel Arends, I.W.C.E.; Hanefeld, U. Development of Screening Methods for Transketolase Activity and Substrate Scope. Tetrahedron Lett. 2012, 7, 790-793. (27) (a) Horii, S.; Fukase, H.; Matsuo, T.; Kameda, Y.; Asano, N.; and Matsui, K. Synthesis and Alpha-D-Glucosidase Inhibitory Activity of N-Substituted Valiolamine Derivatives as Potential Oral Antidiabetic Agents. J. Med. Chem. 1986, 6, 10381046. (b) Hricovíniová Z. Isomerization as a Route to Rare Ketoses: the Beneficial Effect of Microwave Irradiation on Mo(VI)-Catalyzed Stereospecific Rearrangement. Tetrahedron: Asymmetry 2008, 19, 204-208. (28) Zhang, Q.; Bartels, D. Physiological Factors Determine the Accumulation of D-Glycero-D-Ido-Octulose (D-G-D-I-oct) in the Desiccation Tolerant Resurrection Plant Craterostigma plantagineum. Funct. Plant. Biol. 2017, 43, 684-694 (29) Zhang, Q.; Bartels, D. Octulose: a Forgotten Metabolite? J. Exp. Bot. 2017, 68, 5689-5694. (30) (a) Littlechild, J.; Turner, N.; Hobbs, G.; Lilly, M.; Rawas, A.; Watson, H. Crystallization and Preliminary X-ray Crystallographic Data with Escherichia coli Transketolase. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1995, 51, 1074-1076. (b) Asztalos, P.; Parthier, C.; Golbik, R.; Kleinschmidt, M.; Hubner, G.; Weiss, M.S.; Friedemann, R.; Wille, G.; Tittmann, K. Strain and Near Attack Conformers in Enzymic Thiamin Catalysis: Xray Crystallographic Snapshots of Bacterial Transketolase in Covalent Complex with Donor Ketoses Xylulose 5-phosphate and Fructose 6-phosphate, and in Noncovalent Complex with Acceptor Aldose Ribose 5-phosphate. Biochemistry 2007, 46, 12037-12052. (c) Nauton, L.; Hélaine, V.; Thery, V.; Hecquet, L. Insights Into the Thiamine Diphosphate Enzyme Activation Mechanism. Biochemistry 2016, 14, 2144-2152. (31) (a) Sundström, M.; Lindqvist, Y.; Schneider, G.; Hellman, U.; Ronne, H. Yeast TKL1 Gene Encodes a Transketolase that is Required for Efficient Glycolysis and Biosynthesis of Aromatic Amino Acids. J. Biol. Chem. 1993, 268, 24346-24352. (b) Nilsson, U.; Meshalkina, L.; Lindqvist, Y.; Schneider, G. Examination of Substrate Binding in Thiamin Diphosphate-Dependent Transketolase by Protein

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Wild-Type Transketolase L382X

H462X

F435X R521X S385X

OH OH O COOH Hydroxypyruvate

O

O +

OH OH

Evolved Transketolases

n

Aldose (C4-6)

HO OH

CO2

OH

n

Ketose (C6-8)

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Scheme 1. Irreversible reaction catalyzed by TKs in the presence of hydroxypyruvate (HPA) as donor substrate with (2R)-hydroxyaldehydes as acceptor substrates. 149x40mm (300 x 300 DPI)

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A)

Specific activity (U.mg-1)

0,6 0,5 0,41

0,4 0,3 0,17

0,2

0,11

0,10

0,1

0,05

0,03

0 L-glyceraldehyde

C3

D-threose

L-erythrose

1 C4

2 C4

B) 1,4 1,21

1,2 Specfic activity (U.mg-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 0,8 0,6 0,4

0,51 0,34

0,33 0,24

0,2

0,15

0,10 0,0010,024

0 D-ribose

3 C5

D-xylose

4 C5

D-glucose

6 C6

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Table 2. Preparative-scale synthesis of ketoses 7-12 from aldoses 1-6 catalyzed by engineered TKgst variants. 148x32mm (300 x 300 DPI)

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TOC 254x190mm (96 x 96 DPI)

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