Stereoselective CC bond formation catalysed by engineered ... - Nature

ARTICLES PUBLISHED ONLINE: 3 APRIL 2011 | DOI: 10.1038/NCHEM.1011

Stereoselective C–C bond formation catalysed by engineered carboxymethylproline synthases Refaat B. Hamed1,2, J. Ruben Gomez-Castellanos1†, Armin Thalhammer1†, Daniel Harding1, Christian Ducho1‡, Timothy D. W. Claridge1 and Christopher J. Schofield1 * The reaction of enol(ate)s with electrophiles is used extensively in organic synthesis for stereoselective C–C bond formation. Protein-based catalysts have had comparatively limited application for the stereoselective formation of C–C bonds of choice via enolate chemistry. We describe protein engineering studies on 5-carboxymethylproline synthases, members of the crotonase superfamily, aimed at enabling stereoselective C–C bond formation leading to N-heterocycles via control of trisubstituted enolate intermediates. Active site substitutions, including at the oxyanion binding site, enable the production of substituted N-heterocycles in high diastereomeric excesses via stereocontrolled enolate formation and reaction. The results reveal the potential of the ubiquitous crotonase superfamily as adaptable catalysts for the control of enolate chemistry.

T

he stereoselective formation of C–C bonds via the reaction of enol(ate)s with electrophiles is of central importance in synthetic organic chemistry. The economic and environmental advantages of biocatalysis have led to a desire to develop biocatalysts for such reactions. Extensive efforts have been made to develop catalytic antibodies for stereoselective carbon–carbon bond formation reactions (such as aldol reactions), and the engineering of various enzyme families, including aldolases, has been reported1–4. To date, however, it has not been possible to generate a biologically based platform methodology for enol(ate) generation that can be efficiently modified for the formation of C–C bonds of choice. One of the largest enzyme families known to use enolate intermediates is the crotonase superfamily5. Despite their widespread occurrence and biological significance, there are few studies about the application of crotonase superfamily enzymes in biocatalysis6,7 or indeed their engineering8,9. Carboxymethylproline synthases (CMPSs, such as CarB from Pectobacterium carotovorum and ThnE from Streptomyces cattleya) are crotonase superfamily enzymes that catalyse an early step in carbapenem antibiotic biosynthesis, the reaction of malonyl–CoA with L-GHP (an equilibrium mixture of L-glutamate semialdehyde, 5-hydroxy-L-proline and L-pyrroline-5carboxylate (1)) to form (2S,5S)-carboxymethylproline (t-CMP) (Fig. 1a), which is subsequently converted to the bicyclic b-lactam nucleus that is characteristic of carbapenems10–14. It is proposed that CMPS catalysis proceeds via decarboxylation of malonyl–CoA to give an enzyme-bound enolate that is stabilized in an ‘oxyanion hole’ (Fig. 1b). The enolate can then react with the imine form of L-GHP (L-P5C, 1) in a C–C bond-forming reaction to give a thioester intermediate that hydrolyses to give t-CMP and CoASH11,12,14. CarB also catalyses the reaction of L-GHP with a C2 epimeric mixture of methylmalonyl–CoA (3) to give a 55:45 mixture of (6R)- and (6S)-epimers of 6-methyl-t-CMP, under standard incubation conditions. However, we found that, under the same conditions, the ThnE-catalysed reaction produced a 80:20 mixture of the same epimers (Fig. 1a; Table 1, entries 1 and 2)10. This result suggested the possibility of engineering CarB/ThnE with the aim of controlling the formation/reactivity of the intermediate trisubstituted enolates and their reactions with electrophiles.

Here, we report studies on CMPS variants aimed at controlling the formation and/or reactivity of trisubstituted enolate intermediates. The results reveal that active site substitutions of CMPSs can enable the catalysis of stereoselective C–C bond-forming reactions to give products in high diastereomeric excesses. The crotonase superfamily may thus serve as a suitable platform for the development of engineered enzymes for the control of enolate formation and/or reactivity.

Results Selection of residues for substitution and screening of the generated variants for the production of 6-methyl-t-CMP epimers. Initially, we examined the sequences of known and putative CMPSs10 (Fig. 1c) in the light of a crystal structure for CarB12 to identify candidate residues for substitutions that may enable the stereoselective conversion of C2 epimeric methylmalonyl– CoA (3) to (6R)- or (6S)-epimers of 6-methyl-t-CMP. Although crystal structures of CarB/ThnE in complex with L-GHP are not available, knowledge that the enolate intermediate is probably bound in an ‘oxyanion hole’ formed by the backbone amide NHs of Met108CarB/Val153ThnE and Gly62CarB/Gly107ThnE10,12, analogous to many members of the crotonase superfamily5, enabled docking studies that identified residues likely to interact with the substrates. Following initial studies and structural analyses, three residues were selected for CarB substitutions. One of these was the oxyanion hole forming residue Met108, which was substituted for a valine residue, as observed for the analogous residue in ThnE, Val153 (Fig. 1c). Two other residues, Gln111 and Trp79, were selected because they are proposed to be involved in L-GHP binding and to form part of the hydrophobic face of the active site, respectively (Fig. 1b). Screening of the CarB Met108Val, Gln111Asn and Trp79Phe variants by analytical liquid chromatography mass spectrometry (LC-MS) for the formation of 6-methyl-t-CMP from L-GHP and C2 epimeric methylmalonyl–CoA (3) revealed that they formed the C6 epimers of 6-methyl-t-CMP in 95:5, 70:30 and 17:83 ratios of (6R):(6S) epimers, respectively, under standard incubation conditions. For each of these variants, the reactions were scaled up, and the stereochemical assignments (Supplementary Figs S2 and S3) and diastereomeric ratios (d.r.) of the two epimers were

1

Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK, 2 Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, 71256, Egypt; † These authors contributed equally to this work; ‡ Present address: Department of Chemistry, Institute of Organic and Biomolecular Chemistry, Georg-August-University Go¨ttingen, Go¨ttingen, Germany. * e-mail: christopher.schofi[email protected] NATURE CHEMISTRY | VOL 3 | MAY 2011 | www.nature.com/naturechemistry

© 2011 Macmillan Publishers Limited. All rights reserved.

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ARTICLES a

NATURE CHEMISTRY 'Oxy-anion hole' O

O

CoAS

OH

+

b

N H +

Q111 COO–

R Alkylmalonyl-CoA

M108

O

CO2

R

H

O

6S

5 OH HN 2

+ COOH

R = CH3: CarB R = CH3: ThnE

W79 L-P5C

Oxy-anion hole

CoASH R

V140

Imine form (L-P5C)

CarB/ThnE

c

DOI: 10.1038/NCHEM.1011

H229

G62 H

6R OH HN COOH

Malonyl-CoA

# E131

55(6R):45(6S) 80(6R):20(6S)

*

*

#

CarB: 61 GGDFNEVKQLSRSEDIEEWIDRVIDLYQAVLNVNKPTIAAVDGYAIGMGFQFALMFDQRLMASTANFVMPE131 ThnE: 106 GGDFHEVSEFTGGDEVNAWIDDITDLYTTVAAISKPVIAAIDGYAIGVGLQISLCCDYRLGSEQARLVMPE 176

Figure 1 | CarB and ThnE are crotonase superfamily enzymes catalysing enolate alkylation. a, Formation of the C6 epimers of 6-alkyl-t-CMP from C2 epimeric alkylmalonyl–CoA and pyrroline-5-carboxylate (L-P5C, 1) by CMPS catalysis. The imine form (L-P5C, 1) of the substrate exists in equilibrium with the cyclic hemiaminal and aldehyde/hydrated aldehyde forms11,14. b, View derived from a crystal structure of CarB12 with malonyl–CoA and L-P5C (1) modelled into the active site. c, Partial sequence alignment of CarB (P. carotovorum) and ThnE (S. cattleya)10 with identical and similar residues highlighted. The proposed oxyanion hole forming residues are marked with * and the catalytically important (at least for CarB) glutamate residue with #. The figure was generated using Pymol (www.pymol.org), Clustal W22 and Genedoc (http://www.nrbsc.org/gfx/genedoc/).

confirmed by nuclear magnetic resonance (NMR) analyses (Supplementary Fig. S4; Table 1, entries 3–5). These results demonstrate that active site substitutions can make a substantial difference to the stereochemical outcome of CMPS catalysis. The results for the CarB M108V and CarB W79F variants were encouraging, because the d.r. values obtained were substantially different from wild-type CarB and implied that it may be possible to obtain highly stereoselective formation of either (6R)- or (6S)-6-methyl-t-CMP ((2S,5S,6R)-6 or (2S,5S,6S)-6). The results stimulated a second round of substitutions involving the evaluation of further variants at Met108CarB and/or Trp79CarB: CarB M108I, CarB M108L, CarB M108A, CarB M108A/W79F (double variant), CarB M108V/W79F (double variant) and CarB W79A, as well as CarB H229A. These variants formed the two C6 epimers of 6-methyl-tCMP in 92:8, 47:53, 45:55, 11:89, 56:44, 16:84 and 75:25 ratios of (6R):(6S) epimers, respectively (Supplementary Fig. S4; Table 1, entries 6–12). The ‘isolated’ yields of the C6 epimers of 6-methylt-CMP, produced from C2 epimeric methylmalonyl–CoA (3) and L-GHP by different CMPS catalysis, were determined by a combination of LC-MS and 1H NMR analyses (Table 1); the same method was used subsequently to quantify other substituted N-heterocycles produced by CMPS catalysis (Table 1). Although some of the isolated yields were lower than those for wild-type CarB, in some cases (for example, CarB W79F and CarB M108I) highly stereoselective CMPS variants produced 6-methyl-t-CMP epimers in a comparable or higher yield than the wild-type enzymes. These results reveal that single substitutions at the active site of CarB can substantially vary the d.r. of the product from the 55:45 (6R:6S) ratio obtained for the wild-type CarB, and that significant bias can be achieved towards either the (6R)-6-methyl-t-CMP ((2S,5S,6R)-6) epimer (for example, CarB M108V and CarB M108I) or the (6S)-6-methyl-t-CMP ((2S,5S,6S)-6) epimer (for example, CarB M108A/W79F). 366

We then carried out substitutions on wild-type ThnE in an analogous manner to wild-type CarB. The following ThnE variants were prepared and evaluated for the formation of the C6 epimers of 6-methyl-t-CMP (6R:6S): ThnE V153I (78:22), ThnE V153M (13:87), ThnE V153L (10:90), ThnE V153A (9:91), ThnE W124F (55:45) and ThnE V153M/W79F, double variant (13:87) (Supplementary Fig. S5; Table 1, entries 13–18). These results demonstrate that, as for CarB, ThnE is amenable to residue substitution to alter its stereoselectivity. The yield of the stereoselective variant ThnE V153M (Table 1, entry 14) was 12 times higher than that of wild-type, revealing that substitution can improve yield as well as stereoselectivity. Screening of variants for the production of 6-ethyl-t-CMP epimers. We then investigated whether the CMPS variants can catalyse the stereoselective formation of trisubstituted enolates with alkyl groups other than methyl by testing the reaction of L-GHP with C2 epimeric ethylmalonyl–CoA (4)10. Although none of the ThnE variants tested was able to produce isolable quantities of 6-ethyl-t-CMP epimers, some of the CarB variants catalysed the production of the two C6 epimers of 6-ethyl-t-CMP10 in reasonable yields, as revealed by analytical LC-MS analyses. Subsequent scale-up of the reaction for one of the highest yielding variants (CarB W79F, see below), purification of the products and NMR analyses (Figs 2b and 3a) of the separated epimers enabled assignment of the stereochemistry at C6 of the epimer with shorter retention time as (6R)- (that is, (2S,5S,6R)-7) and that of the latter eluting epimer as (6S)-6-ethyl-t-CMP ((2S,5S,6S)-7) (Supplementary Figs S6 and S7). To confirm the stereochemical assignments at C6 of the two epimers of 6-ethyl-t-CMP, the (2S,5S,6R)-6-ethylcarbapenam methyl ester was synthesized from protected (2S)-glutamate and its structure was confirmed by NMR analyses (Supplementary Scheme S1 and Fig. S13). Enzyme-mediated NATURE CHEMISTRY | VOL 3 | MAY 2011 | www.nature.com/naturechemistry


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Table 1 | The N-heterocycles resulting from incubation of C2 epimeric alkylmalonyl–CoA and L-GHP/L-AASA (2) with CMPS. O

O

+ CoAS

OH

H

( )n

N

R

OH

O

A Entry 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

Substrate A (R) CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 C2H5 C2H5 C2H5 C2H5 C2H5 C2H5 CH3 CH3 CH3 CH3 CH3 CH3 C2H5 C2H5 C2H5 C2H5

Carboxymethylproline synthase

R2 H

O HO

HN

Wild-type CarB Wild-type ThnE CarB M108V CarB Q111N CarB W79F CarB M108I CarB M108L CarB M108A CarBW79F/M108A CarBW79F/M108V CarB W79A CarB H229A ThnE V153I ThnE V153M ThnE V153L ThnE V153A ThnE W124F ThnE V153M/W124F Wild-type CarB CarB M108L CarBW79F CarB W79F/M108A CarB W79F/M108V CarB W79A Wild-type CarB CarB M108L CarB W79F CarB W79F/M108A CarB W79F/M108V CarB W79A CarB W79F CarB W79F/M108A CarB W79F/M108V CarB W79A

( )n COOH

B Enzyme

B (n) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2

- CO2 - CoASH

R1

Product n 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2

R1 CH3 CH3 CH3 CH3 H CH3 CH3 H H CH3 H CH3 CH3 H H H CH3 H C2H5 C2H5 H H H H CH3 CH3 H H H H H H H H

R2 H H H H CH3 H H CH3 CH3 H CH3 H H CH3 CH3 CH3 H CH3 H H C2H5 C2H5 C2H5 C2H5 H H CH3 CH3 CH3 CH3 C2H5 C2H5 C2H5 C2H5

d.r. (R:S) 55:45 80:20 95:5 70:30 17:83 92:8 47:53 45:55 11:89 56:44 16:84 75:25 78:22 13:87 10:90 9:91 55:45 13:87 65:35 72:28 32:68 17:83 34:66 12:88 66:34 60:40 17:83 5:95 24:76 1:99 13:87 9:91 19:81 ,1:99*

Yield (%) 34 4 14 21 55 24 49 43 19 26 34 51 8 48 39 25 3 13 4 5 44 12 12 36 7 5 9 12 10 11 4 2 1 6

R1 and R2 represent the substituents of the major epimer produced. d.r. refers to C6 (t-CMP)/C7 (t-CMPi) position. Variants that give d.r. ≥ 9:1 have their d.r. values highlighted in bold. *The (7R)-7-ethyl-t-CMPi ((2S,6S,7R)-9) epimer was not detected.

ester and b-lactam hydrolysis of the prepared carbapenam gave (6R)-6-ethyl-t-CMP, which has the same retention time as the product assigned as (6R)- prepared by CarB W79F catalysis (Supplementary Fig. S8). Co-chromatography of (6R)-6-ethyl-tCMP produced by hydrolysis of (2S,5S,6R)-6-ethylcarbapenam methyl ester resulted in enrichment of the peak corresponding to (6R)-6-ethyl-t-CMP ((2S,5S,6R)-7) produced by CarB W79F catalysis (Supplementary Fig. S8). The d.r. values of the two 6-ethyl-t-CMP epimers (6R:6S) produced by CMPS catalysis, under standard conditions, were determined by analytical LC-MS analyses (Fig. 3a) as follows: wildtype CarB (65:35), CarB M108L (72:28), CarB W79F (32:68), CarB M108A/W79F (17:83), CarB M108V/W79F (34:66) and CarB W79A (12:88) (Table 1, entries 19–24). CarB Met108V and CarB M108I produced the two epimers of 6-ethyl-t-CMP with a d.r. of 80:20, but the yields were low (,2%). With C2 epimeric ethylmalonyl–CoA (4) as a substrate, the CarB W79F and CarB W79A variants maintained the bias towards the (6S)-6-alkyl-t-CMP epimer that was observed with methylmalonyl–CoA (3). Notably higher yields of 6-ethyl-t-CMP than wild-type CarB (10× higher; Table 1, entries 21 and 24) were observed. Although the observed stereoselectivities

for the production of 6-ethyl-t-CMP epimers were lower than those for the 6-methyl-t-CMP epimers, these results demonstrate that t-CMP derivatives with different C6 alkyl substituents can be produced by CMPS variant catalysis. Screening of variants for the production of 7-methyl- and 7-ethyl-t-CMPi epimers. CarB is reported to catalyse the production of N-heterocycles with ring sizes other than five7. We therefore investigated whether CMPS variants can catalyse the stereoselective formation of the C7 epimers of 7-methyl(2S,6S)-carboxymethylpipecolic acid (7-methyl-t-CMPi) from L-aminoadipate semialdehyde (L-AASA (2))7 and C2 epimeric methylmalonyl–CoA (3) (Table 1, n ¼ 2). Although the tested ThnE variants did not catalyse the formation of detectable levels of 7-methyl-t-CMPi, some of the CarB variants did (Figs 3c and 2c). The d.r. values of the two epimers (7R:7S) were as follows: wild-type CarB (66:34), CarB M108L (60:40), CarB W79F (17:83), CarB M108A/W79F (5:95), CarB M108V/W79F (24:76) and CarB W79A (1:99) (Fig. 3c; Table 1, entries 25–30). The CarB W79-based, but not other, variants catalysed the production of the two C7 epimers of 7-ethyl-t-CMPi from L-AASA (2) and C2

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9

H2

H7(S)

CarB W79A d.e.= 0.98

H6

H H'

H

7

H9

H5' H4'

H5 H3' H4

H3

8

H' H H OH HN 2 H' H

O

H8

d

H

6

COOH (7S)-7-ethyl-t-CMPi

O

c H2

b

H6

H7(S) H3


H5' H4'

H5 H3' H4

H8


H3

H4

H3'

H

H H'

H' H H OH HN 2 H' H 7

6

COOH

H2 H5

8

(7S)-7-methyl-t-CMPi

O

H6(S)

H

H4'

H8

H7 H7'

H

8

7

H'

H 6

HO

H

5

HN 2

H H' H COOH

(6S)-6-ethyl-t-CMP

O

H2 H6(R)

CarB M108L d.e.= 0.44

H5

H3

H4

7

H8

8

H 6

H7 H7' H3'

H

HO

H' H

5 2

HN

H4'

H H'

H COOH

(6R)-6-ethyl-t-CMP

a

O

H

7

H 6

HO

6S

ThnE V153A d.e.= 0.82 6R

H5

4.0

3.5

3.0

H

HO

6S

H H' H COOH

7

H

H' H

5

HN

2

H H'

H COOH

(6R)-6-methyl-t-CMP

H6(R)

CarB d.e.= 0.10

HN 2

6

6R

H2

H

(6S)-6-methyl-t-CMP

O

CarB M108V d.e.= 0.90

H'

5

H3 H6(S)

2.5

H4

H3'

2.0

H4'

H7

1.5

1.0

ppm

Figure 2 | 1H NMR spectra for purified products of CMPS catalysis, produced under standard conditions, showing results for variants displaying a high degree of diastereoselectivity. a, Mixtures of 6-methyl-t-CMP epimers produced by incubation of C2 epimeric methylmalonyl–CoA (3) and L-GHP with the shown CMPSs. b, Separated 6-ethyl-t-CMP epimers produced by incubation of C2 epimeric ethylmalonyl–CoA (4) and L-GHP with the shown CMPSs. c, 7-Methyl-t-CMPi epimer observed upon incubation of C2 epimeric methylmalonyl–CoA (3) and L-aminoadipate semialdehyde (L-AASA (2)) with CarB W79A. d, 7-Ethyl-t-CMPi epimers observed upon incubation of C2 epimeric ethylmalonyl–CoA (4) and L-AASA (2) with CarB W79A.

epimeric ethylmalonyl–CoA (4) (Fig. 2d; Supplementary Fig. S10b). The d.r. values of the two 7-ethyl-t-CMPi epimers (7R:7S) were as follows: CarB W79F (13:87), CarB M108A/W79F (9:91), CarB M108V/W79F (19:81) and CarB W79A (,1:99) (Table 1, entries 31–34). NMR analyses (Fig. 2d; Supplementary Figs S10a and S11a) of the major epimer produced by CarB W79A catalysis enabled assignment of its C7 stereochemistry as (7S). The very high d.r. in the latter case may reflect increasing steric demand for the formation of the six-membered ring product compared to the five-membered one (the d.r. for 6-ethyl-t-CMP by CarB W79A was 12(6R):88(6S); Table 1, entry 24). Effect of pH/temperature on diastereoselectivity. We then investigated the effect of conditions on stereoselectivity of the catalysed reactions using three CMPSs, two of which are selective for the preparation of (6R)-6-methyl-t-CMP ((2S,5S,6R)-6) (CarB and CarB M108V) and one for (6S)-6-methyl-t-CMP ((2S,5S,6S)-6) (ThnE V153A) (Table 1, entries 1, 3 and 16, respectively). Timecourse (Supplementary Fig. S14) analyses at different temperature and pH values revealed that, in each case, the observed diastereoselectivity towards the major epimer produced was enhanced both at early time 368

points and at lower pH or temperature values. These results suggest that a factor in determining the overall diastereoselectivity of the reaction is the rate of equilibration of the C2 epimers of alkylmalonyl– CoA, which occurs because of the acidity of the C2 malonyl hydrogen. Coupling of crotonyl–CoA carboxylase reductase and CMPSs catalyses. To further investigate the diastereoselectivity of CMPScatalysed reactions, we used crotonyl–CoA carboxylase reductase (Ccr) from Rhodobacter sphaeroides to prepare ethylmalonyl–CoA as a single epimer. Ccr catalyses the stereoselective reductive carboxylation of (E)-crotonyl–CoA to give a product that has been assigned as (2S)-ethylmalonyl–CoA15,16 (Fig. 4). Ccr was obtained as described previously15 and used to prepare (2S)ethylmalonyl–CoA, which was directly incubated with L-GHP and CarB W79F (the highest yielding 6-ethyl-t-CMP producer), under standard conditions. Notably, in contrast to the incubation of CarB W79F with epimeric ethylmalonyl–CoA (4), where a 32:68 ratio of (6R):(6S)-6-ethyl-t-CMP was observed (Table 1, entry 21), in the Ccr-CarB W79F coupled reaction a single peak (m/z ¼ 202 [M þ H]þ) corresponding to (6R)-6-ethyl-t-CMP ((2S,5S,6R)-7) was observed (Supplementary Fig. S12c). NATURE CHEMISTRY | VOL 3 | MAY 2011 | www.nature.com/naturechemistry


NATURE CHEMISTRY a O 6R

H 6

HO

Relative intensity 100

c

b

H

O 6S

H

O 6R 6

5

HN 2

HO

COOH

CarB W79A

HN

HO COOH

6S

6S CarB W79F/M108A

5

HO

HN 2

Relative intensity 100

6R

6S

0 100

6S

1h

0 100

6S

30 min

6S

10 min

6S

1 min

6R 6S 6R

100 CarB 0

5.00

6S

10.00 15.00 20.00 Time (min)

25.00

0

5.00

7S 7R

CarB M108L

7R 7S

CarB

7R 7S

0

10.00 15.00 20.00 25.00 Time (min)

7R

CarB W79F

0 100

6R

100

7R 7S

0 100

6R

100

Standard 7S

CarB W79F/M108A

6R

100

COOH

CarB W79A 2.5 h

6R

6R

CarB M108L

Ccr incubation Relative intensity time 5h 100

6S

H

OH HN

COOH

6R

100

O 7S

6

COOH

COOH

100

6S

H 7

OH HN 2

HN

6R

100 0 100

O 7R

H

O 6S

6R

100

CarB W79F

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DOI: 10.1038/NCHEM.1011

5.00

10.00 15.00 20.00 25.00 Time (min)

Figure 3 | Analyses of stereoselective CMPS-catalysed reactions. a, Production of the C6 epimers of 6-ethyl-t-CMP from C2 epimeric ethylmalonyl–CoA (4) and L-GHP by the shown CMPSs. b, Products of the Crotonyl–CoA carboxylase reductase (Ccr)–CarB W79F coupled reaction. The Ccr assay was carried out for the indicated time before addition of the CarB W79F assay components. Note that the longer the time of the initial Ccr reaction, the higher the amount of (6S)-6-ethyl-t-CMP ((2S,5S,6S)-7) produced after subsequent CarB W79F incubation. c, Production of the C7 epimers of 7-methyl-t-CMPi from C2 epimeric methylmalonyl–CoA (3) and L-AASA (2) by the shown CMPSs. The figures show LC–MS extracted ion chromatograms for the product of interest using p-aminosalicylic acid as an internal standard (not shown with a and b for clarity). Ccr-incubation prior to addition of CarB W79F O

O

Ccr CoA-S

L-GHP + CarB W79F

O 2S

CoA-S

OH

NADPH NADP + CO2/H+ (E)-Crotonyl-CoA (2S)-ethylmalonyl-CoA

CO2

OH

(6R)-6-ethyl-t-CMP (Only observed product with short Ccr-incubation time)

O CoA-S

HN

CoASH O

Non-enzymatic epimerization

Preferred CarB W79F substrate

6R

HO

+

H

O

L-GHP + CarB W79F

O 2R

OH

H

O 6S

HO

HN

CO2 CoASH

(2R)-ethylmalonyl-CoA

O

OH

(6S)-6-ethyl-t-CMP

Figure 4 | Crotonyl–CoA carboxylase reductase (Ccr)–CMPS coupled incubations. Stereoselective formation of (6R)-6-ethyl-t-CMP ((2S,5S,6R)-7) from (E)-crotonyl–CoA and L-GHP is observed in Ccr-CarB W79F coupled assays. Both (6R) ((2S,5S,6R)-7) and (6S)-6-ethyl-t-CMP ((2S,5S,6S)-7) are observed when the Ccr incubation time is sufficiently long (before the addition of CarB W79F), probably due to epimerization of the ethylmalonyl–CoA product of Ccr catalysis.

Experiments in which the incubation time with Ccr (before addition of the CarB W79F assay components) was varied were then carried out (Fig. 3b). The results revealed that extending the Ccr incubation time, before addition of CarB W79F, partially restored the bias towards the (6S)-6-ethyl-t-CMP ((2S,5S,6S)-7) epimer that is observed as the major product when using C2 epimeric ethylmalonyl–CoA (4). The most likely explanation for these observations is that the single epimer of ethylmalonyl–CoA (assigned by Alber and colleagues as the (2S) epimer16) produced by Ccr catalysis undergoes C2 epimerization (Fig. 4). Assuming correct assignment of the Ccr product16, these results imply that (2S)-ethylmalonyl–CoA gives rise to (6R)-6-ethyl-t-CMP ((2S,5S,6R)-7), and (2R)-ethylmalonyl-CoA gives rise to (6S)-6-ethyl-t-CMP ((2S,5S,6S)-7) (Fig. 5).

Unlike the incubation of Ccr-produced ethylmalonyl–CoA and with CMPSs, which resulted in the formation of (6R)-6ethyl-t-CMP ((2S,5S,6R)-7) (Supplementary Fig. S12a–c), we found that incubation of Ccr-produced ethylmalonyl–CoA with L-AASA (2) and CarB W79A (the highest yielding 7-ethyl-t-CMPi producer; Table 1, entry 34), under standard conditions, did not result in production of detectable 7-ethyl-t-CMPi (Supplementary Fig. S12e). This result is consistent with the outcome when using C2 epimeric ethylmalonyl–CoA (4), where CarB W79A produced solely (7S)-7-ethyl-t-CMPi ((2S,6S,7S)-9) (Supplementary Fig. S12f, Table 1: entry 34). Taken together, the Ccr–CMPS coupled reactions support a mechanism in which a specific alkylmalonyl–CoA C2 epimer is converted to a specific enolate (Fig. 5). L-GHP



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Figure 5 | Proposed mechanism for the CMPS-catalysed formation and reaction of enolates. The decarboxylation of a specific alkylmalonyl–CoA enantiomer is proposed to give rise to either (E)- or (Z)-enolate, which reacts with the imine form of L-GHP/L-AASA (2) to give a specific diastereomer of carboxymethyl-substituted N-heterocycles. The possibility of limited conversion of (2R)-alkylmalonyl to the (Z)-enolate, and (2S)-alkylmalonyl to the (E)-enolate, cannot be eliminated. For a model for the possible modes of binding of alkylmalonyl–CoA to CMPS, see Supplementary Fig. S15.

Discussion A prerequisite for the stereoselective formation of a C–C bond via an a-substituted enolate intermediate under kinetic control is the selective formation of (E)- or (Z)-enolates. We investigated whether structurally informed generation of specific active site variants of the crotonase superfamily enzymes CarB and ThnE could enable the stereoselective formation of (E)- or (Z)-enolates and their reaction with electrophiles (L-GHP and L-AASA (2)). Following initial studies, we found that substitution of two residues in CarB and subsequently in ThnE, one of which (Met108CarB/Val153ThnE) is involved in forming the oxyanion hole and one of which (Trp79CarB /Trp124ThnE) forms a part of the hydrophobic face of the active site, can significantly affect the observed d.r. of products of CMPS catalysis. Even when using a C2 epimeric mixture of methylmalonyl–CoA (3), single or double substitutions at these positions substantially bias the d.r. of 6-methyl-t-CMP products away from the 55:45 (6R:6S) ratio observed for wild-type CarB. Thus, for example, the CarB M108V variant gives a (6R):(6S) 6-methyl-t-CMP product ratio of ≥95:5, whereas the CarB M108A/W79F variant gives a ratio of ≤11:89 in favour of the (6S) product. The results were extended to the CarB homologue ThnE, ethylmalonyl–CoA (4), and to the L-GHP analogue L-AASA (2). The ThnE variants results suggest that the crotonase superfamily may be generally amenable to variation with a view to producing diastereoselective biocatalysts. As for CarB, single substitutions of ThnE were sufficient to substantially alter the d.r. of the observed products (for example, the ThnE V153A gives a (6R):(6S) 6-methyl-t-CMP product ratio of ≤9:91, whereas the wild-type ThnE gives a 80:20 ratio). Significant variations in d.r. could also be obtained for the CMPS-catalysed reaction of C2 epimeric ethylmalonyl–CoA (4) and L-GHP (for example, Table 1, entry 24) and the reaction of methyl (3)/ethylmalonyl–CoA (4) with L-AASA (2) (for example, Table 1, entries 30 and 34, respectively). Active site variations can also alter the yields obtained; for example, substitution of Trp79CarB for Phe or Ala residues resulted in a substantial (10-fold) enhancement in the overall yield of 6-ethyl-t-CMP compared to wild-type CarB (Table 1, entries 21 and 24). Substitution of wild-type V153ThnE for a Met residue resulted in a significant enhancement in both yield and diastereoselectivity (Table 1, entry 14). We also observed that variations in incubation conditions can further enhance the d.r. values towards the major epimer observed 370

(Supplementary Fig. S14), probably (at least in part) due to differences in the rate of C2 alkylmalonyl–CoA epimerization. These results reveal the potential of CMPS enzymes for dynamic kinetic discrimination based on the acid/base-catalysed equilibration of C2 epimeric alkylmalonyl–CoA substrates. Consistent with this proposal, we found that the use of a single substrate epimer, that is, (2S)-ethylmalonyl–CoA, produced by Ccr-catalysis, can significantly alter the stereochemical outcome of the CMPS-catalysed reaction (Supplementary Fig. S12). These results might be exploited in practical applications using CMPS variants in cells, as they imply that variations in, for example, levels of coupled enzymes ratios may be used to control the diastereoselective formation of the desired product. It is possible that application of other protein engineering approaches, such as directed evolutionary techniques17–19, will also enable enhancement in stereoselectivity and/or expansion in the range of electrophiles accepted in CMPS-catalysed reactions. At present, the precise chemical basis behind the diastereoselectivity exhibited by engineered CMPSs is not clear. The introduced substitutions may influence the binding of alkylmalonyl–CoA, the rate of decarboxylation and/or the rate of L-P5C (1) reaction with the enolate intermediate. The necessary close spatial relationship of malonyl–CoA and L-P5C (1) binding sites implies that some of the targeted residues (for example, Trp79CarB) are probably involved in both decarboxylation and alkylation steps. Several structure– activity relationships can, however, be identified from the results. One notable case is that CarB/ThnE variants with a b-branched residue (that is, Val or Ile) at the oxyanion hole forming residue 108CarB (wild-type Met)/153ThnE (wild-type Val) favour the formation of products with the (6R)-stereochemistry (Table 1, entries 2, 3, 6 and 13). In support of this analysis, for some ThnE variants, substitution of the wild-type Val153 for a non-b-branched residue (Ala, Leu, Met) results in a shift of bias towards the (6S)-epimer of 6-methyl-t-CMP (Table 1, entries 14–16). A steric clash between the alkyl-substituent on the (E)-enolate and b-branched residues at position 108CarB/153ThnE (Supplementary Fig. S15a) provides a possible explanation for these selectivities. Although the CarB/ThnE variants with a b-branched residue at position 108CarB/153ThnE favour the formation of epimers with (6R)-stereochemistry, the Trp79CarB-based variants generally favour the formation of epimers with (6S)/(7S)-stereochemistry (Table 1, entries 8–10, 21–24 and 27–34). The stereoselectivity of NATURE CHEMISTRY | VOL 3 | MAY 2011 | www.nature.com/naturechemistry


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CarB W79F towards products with (6S)/(7S) stereochemistry was further enhanced by introducing an Ala residue at position 108CarB (Table 1, entries 9, 22, 28 and 32) demonstrating the beneficial effects of combining substitutions and that variation of more than one residue can be useful for altering product d.r. The observation that the nascent product of Ccr catalysis gives exclusively the (6R)-6-ethyl-t-CMP ((2S,5S,6R)-7) epimer not only demonstrates the potential of CMPS variants for stereospecific catalysis, but also has mechanistic implications. We propose that decarboxylation of C2 alkylmalonyl–CoA occurs from a stereoelectronically favoured arrangement in which the C2 malonyl-carboxylate is held orthogonally to the CoA–thioester carbonyl group (Supplementary Fig. S15). Assuming the assignment of the Ccr product as (2S)-ethylmalonyl-CoA15,16, the results of Ccr–CMPS coupled incubations show that the decarboxylation of the (2S)epimer of ethylmalonyl–CoA gives rise to the (Z)-enolate (Supplementary Fig. S15b), which reacts with L-P5C (1) to give the (6R)-6-ethyl-t-CMP ((2S,5S,6R)-7) epimer; thus, the (E)-enolate gives rise to the (6S)-6-ethyl-t-CMP ((2S,5S,6S)-7) epimer (Fig. 5). The Ccr–CMPS results also reveal that ‘matching’ of an alkylmalonyl–CoA epimer with an electrophile (L-GHP/ L-AASA, 2) can significantly affect the observed d.r. This is clearly observed in case of the reaction of ethylmalonyl–CoA and L-AASA (2) as catalysed by CarB W79A. Thus, epimeric ethylmalonyl–CoA produces solely (7S)-7-ethyl-t-CMPi ((2S,6S,7S)-9), via reaction of (2R)-ethylmalonyl-CoA, whereas incubation of (2S)ethylmalonyl–CoA (the nascent product of Ccr catalysis) and L-AASA (2) with the same variant results in no detectable production of 7-ethyl-t-CMPi. Overall, our results demonstrate the ability of the crotonase superfamily enzymes to control the formation of trisubstituted enolates and their subsequent reaction with electrophiles to give products in high diastereomeric excess. Given that the crotonase superfamily catalyses a wide range of enolate-based chemistry5,20 involving reactions with a wide range of electrophiles including oxygen21, the results suggest that crotonases may be a useful platform technology for engineering studies aimed at performing enolate reactions of choice.

Methods Preparation of CarB and ThnE variants. Mutagenesis of the plasmid-bearing carB or thnE gene was performed according to the QuikChange Site-Directed Mutagenesis Protocol (Stratagene). Supplementary Tables S1 and S2 give the oligonucleotide primers used for carB and thnE variant preparation, respectively. For details see Supplementary Information. CMPS assays. Small- and large-scale assays of CMPSs and Ccr–CMPS coupled assays were performed and analysed as described in the Supplementary Information. Quantification of yields and d.r. of the products of CMPS catalysis. Yields of different products of CMPS catalysis were calculated using a combination of LC-MS and 1H NMR spectroscopy as follows: (i) for high-yielding reactions the isolated yield, based on protected L-GHP, was quantified (in duplicate) according to a reported 1H NMR method7 using [2H]4-trimethylsilylpropionate as an external standard; (ii) using this 1H NMR quantified yield as a reference, the yields of other CMPSs were determined (mean of two biological repeats) by LC-MS assays using p-aminosalicylic acid as an internal standard. The d.r. of the products resulting from incubation of C2 epimeric (m)ethylmalonyl–CoA and L-GHP/L-AASA (2) by CMPS catalysis were determined by 1H NMR analysis (Supplementary Figs S4 and S5).

Received 1 November 2010; accepted 14 February 2011; published online 3 April 2011

References 1. Schultz, P. & Lerner, R. From molecular diversity to catalysis: lessons from the immune system. Science 269, 1835–1842 (1995). 2. Reymond, J. Stereoselectivity of aldolase catalytic antibodies. J. Mol. Catal. B 5, 331–337 (1998). 3. Dean, S., Greenberg, W. & Wong, C. Recent advances in aldolase-catalyzed asymmetric synthesis. Adv. Synth. Catal. 349, 1308–1320 (2007). 4. Bolt, A., Berry, A. & Nelson, A. Directed evolution of aldolases for exploitation in synthetic organic chemistry. Arch. Biochem. Biophys. 474, 318–330 (2008).

5. Hamed, R. B., Batchelar, E. T., Clifton, I. J. & Schofield, C. J. Mechanisms and structures of crotonase superfamily enzymes—how nature controls enolate and oxyanion reactivity. Cell. Mol. Life Sci. 65, 2507–2527 (2008). 6. Grogan, G., Roberts, G. A., Bougioukou, D., Turner, N. J. & Flitsch, S. L. The desymmetrization of bicyclic b-diketones by an enzymatic retro-Claisen reaction. A new reaction of the crotonase superfamily. J. Biol. Chem. 276, 12565–12572 (2001). 7. Hamed, R. B., Mecinovic, J., Ducho, C., Claridge, T. D. W. & Schofield, C. J. Carboxymethylproline synthase catalysed syntheses of functionalised N-heterocycles. Chem. Commun. 46, 1413–1415 (2010). 8. Yu, W. et al. Mutation of Lys242 allows D3,D2-enoyl-CoA isomerase to acquire enoyl-CoA hydratase activity. Biochim. Biophys. Acta 1760, 1874–1883 (2006). 9. Xiang, H., Luo, L., Taylor, K. L. & Dunaway-Mariano, D. Interchange of catalytic activity within the 2-enoyl-coenzyme A hydratase/isomerase superfamily based on a common active site template. Biochemistry 38, 7638–7652 (1999). 10. Hamed, R. B., Batchelar, E. T., Mecinovic´, J., Claridge, T. D. W. & Schofield, C. J. Evidence that thienamycin biosynthesis proceeds via C-5 epimerization: ThnE catalyzes the formation of (2S,5S)-trans-carboxymethylproline. ChemBioChem 10, 246–250 (2009). 11. Batchelar, E. T. et al. Thioester hydrolysis and C–C bond formation by carboxymethylproline synthase from the crotonase superfamily. Angew. Chem. Int. Ed. 47, 9322–9325 (2008). 12. Sleeman, M. C., Sorensen, J. L., Batchelar, E. T., McDonough, M. A. & Schofield, C. J. Structural and mechanistic studies on carboxymethylproline synthase (CarB), a unique member of the crotonase superfamily catalyzing the first step in carbapenem biosynthesis. J. Biol. Chem. 280, 34956–34965 (2005). 13. Sleeman, M. C. & Schofield, C. J. Carboxymethylproline synthase (CarB), an unusual carbon–carbon bond-forming enzyme of the crotonase superfamily involved in carbapenem biosynthesis. J. Biol. Chem. 279, 6730–6736 (2004). 14. Gerratana, B., Arnett, S. O., Stapon, A. & Townsend, C. A. Carboxymethylproline synthase from Pectobacterium carotorova: a multifaceted member of the Crotonase superfamily. Biochemistry 43, 15936–15945 (2004). 15. Erb, T. J. et al. Synthesis of C5-dicarboxylic acids from C2-units involving crotonyl-CoA carboxylase/reductase: The ethylmalonyl-CoA pathway. Proc. Natl Acad. Sci. USA 104, 10631–10636 (2007). 16. Erb, T. J., Brecht, V., Fuchs, G., Muller, M. & Alber, B. E. Carboxylation mechanism and stereochemistry of crotonyl-CoA carboxylase/reductase, a carboxylating enoyl-thioester reductase. Proc. Natl Acad. Sci. USA 106, 8871–8876 (2009). 17. Johannes, T. W. & Zhao, H. Directed evolution of enzymes and biosynthetic pathways. Curr. Opin. Microbiol. 9, 261–267 (2006). 18. Williams, G. J., Nelson, A. S. & Berry, A. Directed evolution of enzymes for biocatalysis and the life sciences. Cell. Mol. Life Sci. 61, 3034–3046 (2004). 19. Reetz, M. T. Laboratory evolution of stereoselective enzymes: a prolific source of catalysts for asymmetric reactions. Angew. Chem. Int. Ed. 50, 138–174 (2011). 20. Holden, H. M., Benning, M. M., Haller, T. & Gerlt, J. A. The crotonase superfamily: divergently related enzymes that catalyze different reactions involving acyl coenzyme A thioesters. Acc. Chem. Res. 34, 145–157 (2001). 21. Widboom, P. F., Fielding, E. N., Liu, Y. & Bruner, S. D. Structural basis for cofactor-independent dioxygenation in vancomycin biosynthesis. Nature 447, 342–345 (2007). 22. Thompson, J. D., Higgins, D. G. & Gibson, T. J. CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (1994).

Acknowledgements The authors acknowledge the Biotechnology and Biological Sciences Research Council, the Ministry of Higher Education (Egypt, R.B.H.), CONACyT and FIDERH (Mexico, R.G.C.), Cancer Research UK (A.T.) and the Deutsche Akademie der Naturforscher Leopoldina (Germany, C.D.) for financial support. The ccr/pET3d construct was a kind gift from B.E. Alber at the Ohio State University.

Author contributions R.B.H. and D.H. identified and prepared CMPSs. R.G.C. prepared Ccr. R.B.H., D.H. and R.G.C. carried out enzyme assays and analysed the results. R.B.H. purified and ran NMR analysis of the products of enzyme catalysis. R.B.H. and T.D.W.C. analysed the NMR data. A.T. synthesized the (2S,5S,6R)-6-ethylcarbapenam methyl ester. C.D. synthesized protected L-AASA (2). R.B.H. and C.J.S. designed the study and wrote the manuscript. All authors commented on the manuscript.

Additional information The authors declare no competing financial interests. Supplementary information and chemical compound information accompany this paper at www.nature.com/ naturechemistry. Reprints and permission information is available online at http://npg.nature. com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to C.J.S.



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