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(Last modification: 13. May 2010)

 

Question: is the Aldol Switch Common to all Aldol Condensations Carried out by Type III PKS?

 

(Note: most of this is taken from a recent publication: Cook et al. (2010), but it is not identical with the text in that publication)

 

A brief overview

   STS-type PKSs carry out a ring-folding via a C2→ C7 aldol condensation, in contrast to the C6→ C1 Claisen condensation performed by CHS-type enzymes.  The mechanisms underlying the different cyclization reactions have been of considerable interest, and a detailed structural/mutagenesis study performed with P. sylvestris STS1 and M. sativa CHS2 by Austin and co-workers (2004) succeeded in generating a functional STS-type enzyme from CHS2 by site-directed mutagenesis.  The primary structural determinants for the different ring-foldings reside within a small displaced loop in STS1 (called the ‘area 2 loop’); it corresponds to positions T131 to M137 in the CHS2 sequence.  In STS1, this region facilitates the formation of a hydrogen-bond network between T132, E192, and S338 (numbering according to M. sativa CHS2); this is absent in CHS2.   This results in the activation of a cryptic thioesterase activity leading to the release of the tetraketide intermediate, thus initiating the decarboxylation/cyclization characteristic of the STS reaction.  This mechanism has been called the ‘aldol switch’. It was successfully introduced into the M. sativa CHS2 enzyme  through the exchange of just 8 amino acids, resulting in the generation of a fully-functional STS-type enzyme (Austin et al., 2004).  The five most critical amino acid exchanges were made within the area 2 loop, while the remaining three exchanges were compensatory; they are located in protein domains referred to as ‘region 1’ and ‘region 3’.  A comparison of the corresponding positions for area 2, as well as other key residues associated with the aldol switch mechanism, is shown below:

 

Region                                          ▼▼▼▼▼▼▼ area 2 loop (131-137)
 Contact with active site cavity                  ••   •             ▼               ▼
 Aldol switch (CHS numbering)                     T132               E192            S338

                                                 ..   .             .               .     

CHS2 Medicago sativa (P30074)             ..IVC TTSGVDM PGA.. ..VCS E VTA.. ..EYGNM S SAC..
STS1 Pinus sylvestris (CAA43165)          ..IFC STTTPDL PGA.. ..ICS E TTA.. ..EYGNM S SAC..
 

Fig. 1. 
Alignment of key sequence motifs underlying the STS-type ring-folding mechanism.
For simplicity, numbering is according to the Medicago sativa chalcone synthase 2 (CHS2).  Positions 131-137 correspond to the displaced area 2 loop that is critical for the formation of the hydrogen bond network formed by T132, E192, and S338 in P. sylvestris stilbene synthase 1 (STS1).  Only three residues in area 2 (positions 132, 133, and 137; closed circles) are in direct contact with the active site cavity.  The five area 2 positions mutated in M. sativa CHS2 to generate a functional STS-type enzyme  are highlighted in red.
 

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A. Sequence alignments

 

Fig. 2 below summarizes an alignment of partial sequences for various microbial and plant type III PKS in the parts important for the aldol switch mechanism. Whenever possible, a CHS- and a STS-type sequence from the same species was included in the alignment to facilitate these comparisons.  For simplification, all numbering is according to the M. sativa CHS2. Deviations from the consensus in positions T132, E192, and S338 are boxed.
The links lead to the comments in the text; the links in the plant names in the text lead to the discussions of the enzymes, if available.

 

Region                                          ▼▼▼▼▼▼▼ area 2 loop (131-137)
 Contact with active site cavity                  ••   •             ▼               ▼
 Aldol switch (CHS numbering)                     T132               E192            S338

                                                 ..   .             .               .     

Dicotyledons                                     ..  .             .               .   

CHS2 Medicago sativa (P30074)             ..IVC TTSGVDM PGA.. ..VCS E VTA.. ..EYGNM S SAC..

CHS Arachis hypogaea (AAO32821)           ..IFC TTSGVDM PGA.. ..VCS E ITA.. ..DYGNM S SAC..
STS3 Arachis hypogaea (P51069)            ..IFC TTSGVAL PGV.. ..VCS E NTA.. ..NYGNM S SAC..

CHS Vitis vinifera (CAA53583)             ..VFC TTSGVDM PGA.. ..VCS E ITA.. ..EYGNM S SAC..
STS Vitis vinifera (P28343)               ..VFC TTSGVEM PGA.. ..VCS E ITV.. ..EYGNM S SAC..

 CHS Sorbus aucuparia (ABB89213)           ..VFC TTSGVDM PGA.. ..VCS E ITA.. ..DYGNM S SAC..
BIS Sorbus aucuparia (ABB89212)           ..IFC TASCVDM PGA.. ..VCA E ITT.. ..EYGNM G APS..

CHS Cannabis sativa (AAL92879)            ..VFC TTSGVDM PGA.. ..VCS E ITA.. ..EYGNM S SAC..
OLS Cannabis sativa (BAG14339)            ..IFT SASTTDM PGA.. ..VCC D IMA.. ..EHGNM S SST..

CHS Hydrangea macrophylla (AAN76184)      ..VFC TTSGVDM PGA.. ..VCS E ITA.. ..DYGNM S SAC..
STCS Hydrangea macrophylla (AAN76183)     ..VFC TTSGVDM PGC.. ..VCS E MTV.. ..EYGNM S SAC..

CHS1 Polygonum cuspidatum (ABK92282)      ..IMC TTSGVDM PGA.. ..VCS E ITA.. ..DYGNM S SAC..
 STS Polygonum cuspidatum (ACC76753)       ..IVC CIAGVDM PGA.. ..VCS E MTP.. ..DYGNM S SAC..
STS1 Rheum tataricum (AAP13782)           ..IVC CIAGVDM PGA.. ..VRS E MTP.. ..DYGNM S SAC..
BAS Rheum palmatum (AAK82824)             ..IVC CLAGVDM PGA.. ..VCS E MTT.. ..DYGNM S SAT..

Monocotyledons

CHS1 Sorghum bicolor (AAD41873)           ..VFC TTSGVDM PGA.. ..VCS E ITA.. ..EYGNM S SAC..
STS1 Sorghum bicolor (AAL49965)           ..VFC TTSGVDM PGA.. ..VCS E ITV.. ..EYGNM S SVC..
ARS1 Sorghum bicolor (XP_002449744)       ..IFS TYSGARA PSG.. ..ACS E LTL.. ..EFGNM S GTT..
ARS2 Sorghum bicolor (XP_002441839)       ..IFS TYSGARA PSG.. ..ACS E LTL.. ..EFGNM S GTT..

CHS Oryza sativa (CAA61955)               ..VFC TTSGVDM PGA.. ..VCS E ITA.. ..EYGNM S SAC..
ARS1 Oryza sativa (XP_476153)             ..IFS TYSGCSA PSA.. ..ACA E LTL.. ..EYGNM S GTT..
ARS2 Oryza sativa (NP_920020)             ..IFS TYSGCRA PSA.. ..ALS E LTL.. ..EFGNM S GAT..
ARS3 Oryza sativa (NP_001064197)          ..IFS TYSGCRA PSA.. ..ACS E LTL.. ..EYGNM S GAT..

CHS3 Bromheadia finlaysoniana (AAB62876)  ..IFC TTSGIDM PGA.. ..VCS E ITA.. ..EYGNM S SAC..
BBS Bromheadia finlaysoniana (CAA10514)   ..VFC TTSGMDL PGA.. ..VCA E TTT.. ..EYGNM S SVC..
BBS Phalaenopsis sp. (CAA56276)           ..IFC TTSGMDL PGA.. ..VCA E TTT.. ..EYGNM S SVC..

Gymnosperms

CHS1 Pinus sylvestris (CAA43166)          ..IFC TTSGVDM PGA.. ..VCS E ITA.. ..DYGNM S SAC..
STS1 Pinus sylvestris (CAA43165)          ..IFC STTTPDL PGA.. ..ICS E TTA.. ..EYGNM S SAC..
CHS1 Pinus strobus (CAA06077)             ..IFC TTSGVDM PGA.. ..VCS E ITA.. ..EYGNM S SAC..
STS1 Pinus strobus (CAA87012)             ..IFC TTTTPDL PGA.. ..VCS E NTA.. ..EYGNM S SAC..
CHS Pinus densiflora (BAA94594)           ..IFC TTSGVDM PGA.. ..VCS E ITA.. ..DYGNM S SAC..
STS Pinus densiflora (BAA94593)           ..IFC STTTPDL PGA.. ..ICS E TTA.. ..EYGNM S SAC..

Fern

CHS Psilotum nudum (BAA87922)             ..VFC TTSGVDM PGA.. ..VCS E ITA.. ..DYGNM S SAC..
STS Psilotum nudum (PnL)(BAA87924)        ..VFC TTGPVS- PGA.. ..VCS E STA.. ..EFGNM S SAT..
STS Psilotum nudum (PnI)(BAA87925)        ..VFC TTAPVTL PGV.. ..VCS E TTA.. ..EFGNM S SAT..

Mosses
STCS1 Marchantia polymorpha (AAW30009)    ..VFA TTSGVNM PGA.. ..VVS E LTC.. ..NYGNM S GAS..
STCS2 Marchantia polymorpha (AAW30010)    ..VMA TTSGVNM PGA.. ..ICS E VTA.. ..DYGNM S SAS..

CHS Physcomitrella patens (ABB84527)      ..VFA TTSGVNM PGA.. ..VAS E VTA.. ..EFGNM S SAS..
ARS Physcomitrella patens (ABU87504)      ..IVF SSTGMLT PAI.. ..VCT E LSS.. ..NKGNM S SAS..

Fungus

ORAS Neurospora crassa (XP_960427)        ..VST TCTDSAN PGY.. ..LAL E VST.. ..NHGNS S SAT..

Bacteria

SrsA Streptomyces griseus (YP_001821984)  ..MFT SVTGIAA PSV.. ..LSV E LCS.. ..DVGNL S SSS..
CHS-LK Synechococcus (CAE07508)           ..VTV SCTGFQS PGV.. ..CAV E LCS.. ..DHGNM S SAT..
 ArsB Azotobacter vinelandii (YP_002800096)..IHV TCSGYLS PSP.. ..HRV D IVH.. ..ENGNM S SAT..
ArsC Azotobacter vinelandii (YP_002800095)..IHV TCSGYLA PSP.. ..TRV D IAH.. ..ENGNM S SST..
 THNS Streptomyces coelicolor (NP_625495)  ..IYV SCTGFMM PSL.. ..VAC E FCS.. ..EYGNI A SAV..

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B. Discussion and comments

 

(Note: the links in 'more' connect to pages discussing these enzymes)

Dicotyledons
  The signature characteristic for the aldol switch-type mechanism is not recognizable in the Arachis hypogaea STS3 and Vitis vinifera STS (more...), but a preliminary analysis of their three-dimensional structures suggested that other residues are responsible for carrying out a similar mechanism in these cases (Austin et al., 2004). 
   The Sorbus aucuparia biphenyl synthase (BIS, more...) and the Cannabis sativa olivetol synthase (OLS, more...) both contain an alanine (A) instead of the aldol switch-type T132 within area 2. Moreover, the BIS sequence has a glycine (G) in place of S338, and OLS has an aspartic acid (D) residue in place of E192.  All three positions play an essential role in the aldol switch hydrogen-bonding network, and the differences suggest that these STS-type enzymes use an alternative aldol condensation cyclization mechanism. This is also likely true in the case of the Rheum tataricum STS1 which contains an isoleucine (I) at position 132, and which is very closely related to a protein from Polygonum cuspidatum (more...) that is annotated as putative STS in the databases.
    One possible candidate for a similar mechanisms was the benzalacetone synthase (BAS) from Rheum palmatum, because the product formation includes the release of a diketide acid that is decarboxylated to the end product benzalacetone (more...). However, the Thr132 in STS is replaced by a Leu in BAS (see above), and thus it seems not possible that an aldol switch like mechanisms plays a role in the BAS reaction.
   The stilbenecarboxylate synthases (STCS) from Hydrangea macrophylla will be discussed in context with the STCSs from the liverwort Marchantia polymorpha, see below.
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Monocotyledons
    The bibenzyl synthase (BBS)-type PKS area 2 sequences from Bromheadia finlaysonia and Phalaenopsis spp. show significant similarities to the A. hypogaea STS3 and V. vinifera STS sequences, suggesting a similar mechanism for these enzymes, i.e. not a typical aldol switch.
   The two Sorghum bicolor  (more...) and three Oryza sativa alkylresorcinol synthases  (more...) are unique with respect to their area 2 sequence: in the position equivalent to T132 in M. sativa CHS2, a tyrosine (Y) is found in all five enzymes. This residue is also discussed in the context of substrate preference for long-chain acyl-CoAs, however its presence in this position also raises questions concerning whether these ARSs possess a STS1-like aldol switch mechanism. The position and size of the tyrosine, as well as the pKα value of its hydroxyl group (pKα ≈ 10, versus 15 for threonine), would suggest that the STS-type cyclization model is not applicable to these enzymes. However, in absence of crystal structures it is not possible to draw definitive conclusions, considering the minor conformational differences observed between CHS- and STS-type enzymes. In this context it is also noteworthy that the STS cloned from Sorghum is identical in the region 2 with the CHS from Sorghum; this indicates that the monocotyledon STS-type enzyme does not use the aldol-switch mechanisms identified with the Gymnosperm enzyme.
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Gymnosperms
   A Gymnosperm STS was the basis for the identification of the aldol switch. Analysis of the STS and CHS-type PKSs from various Pinus species revealed that all the CHS were identical in the residues for area 2, and in the positions 192 and 338.  And all the STS showed the aldol switch signature (with the exception of a S → T exchange in position 131 of the Pinus strobus STS), suggesting a conservation of the aldol switch in STS-type enzymes in gymnosperms. 
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Fern
  For the two STS-type sequences from the primitive fern Psilotum nudum it is difficult to predict the underlying mechanism for the aldol cyclization performed,  solely based on primary sequence data: the STS PnL contains a gap in the area 2 sequence, and both PnL and PnI contain a  unique P134 residue.
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Mosses
   The stilbenecarboxylate synthases (STCS) from Hydrangea macrophylla (more...) and from the liverwort Marchantia polymorpha (more...) are unique with respect to the other type III PKS enzymes, because they use an aldol condensation-type cyclization mechanism but retain the terminal carboxyl group in the end product. Their sequences within area 2 are identical to that of typical CHS-type PKSs, and the three-dimensional structure of the M. polymorpha STCS2 enzyme (PDB: 20UA) revealed no significant differences when compared with that of M. sativa CHS2 within the area 2 loop or other regions associated with aldol switch function.  The M. polymorpha cyclization mechanism must therefore differ from the aldol switch-type; actually it has been proposed that the aldol condensation in resorcylic acid synthases such as STCS is a non-enzymatic reaction of  the released tetraketide pyrone (more...). 
   Like with the fern proteins (see above), it is difficult to predict the mechanism for a putative alkylresorcinol synthase/apyrone synthase (ARS/APS) identified from the moss Physcomitrella patens (more...). 
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Fungus
    The single fungal type III PKS from N. crassa has been alternatively described as an oxoalkylresorcylic acid synthase and an alkylresorcinol synthase by two groups working independently.  Funa and co-workers first identified 2’-oxoalkylresorcylic acids (derived from four condensations) as the major in vitro products, and thus named the enzyme ORAS, whereas Goyal et al. identified 5-alkylresorcinols (derived from three condensations) as the major products (more...). Crystal structures were independently established by two groups (more...), and although their interpretations differ in some details, both studies concluded that an aldol switch-type cyclization mechanism is not involved, due to the presence of a cysteine residue in the position equivalent to T132 in the Medicago sativa CHS2; this would preclude the formation of a thioesterase-like hydrogen bonding network.  The significance of this conclusion, however, could depend on the products identified: alkylresorcinolic products (three condensations, ring-folding like STS) would suggest an alternative mechanism for aldol condensation, whereas alkylresorcylic acid products (four condensations, retention of terminal carboxyl group like with STCS) would not require that.  According to the model previously proposed for resorcylic acid biosynthesis, the formation of such molecules is likely a non-enzymatic reaction from released pyrones  (more...). 
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Bacteria
    Three bacterial type III PKS enzymes have also been shown to posses alkylresorcinol synthase activity: SrsA from Streptomyces griseus (more...), CHS-LK from Synechococcus (more...), and ArsB from Azotobacter vinelandii (more...).  SrsA contains a valine (V) at the critical 132 position of area 2, and the Synechococcus and A. vinelandii enzymes both contain a cysteine (C) residue at this position, as noted above for the N. crassa ORAS enzyme.  Additionally, the otherwise strictly conserved E192 is replaced by aspartic acid in ArsB and ArsC, as was also noted for the C. sativa OLS.  All three bacterial enzymes are therefore unlikely to posses the classical aldol switch mechanism.  Within the Azotobacter ars operon a gene encoding a second type III PKS was also identified (ArsC) which shares 71% amino acid identity with ArsB.  Like ArsB, ArsC was also shown to accept various fatty acyl-CoA starter units, however, in contrast to ArsB, the major in vitro products from ArsC were found to be tetraketide pyrones (more...).  While both enzymes generate an identical tetraketide intermediate from three condensation reactions, cyclization of the ArsC intermediates would be predicted to occur via intramolecular C5 oxygen→C1 lactonization rather than the aldol condensation used by ArsB and other alkylresorcinol synthases.  The diagnostic area 2 sequences found in ArsB and ArsC are identical with the exception of position 137 (S in ArsB; A in ArsC); this is one of the three residues contacting the second subunit in the PKS homodimer. Thus, it is unlikely that this region is responsible for differences in the products produced by these enzymes (i.e., alkylresorcinol versus tetraketide alkylpyrone).
   Of particular interest within the context of cyclization specificity is the enzyme 1,3,6,8-tetrahydroxynaphthalene synthase (product = T4HN, enzyme name THNS or Rppa), a bacterial type III PKS present in various Streptomyces strains (more...). These enzymes perform four condensation reactions resulting in a linear pentaketide intermediate, and use both a Claisen and an aldol condensation for the formation of the end product. The THNS from S. coelicolor has been crystallized, and the analysis unexpectedly revealed that position 106, which corresponds to T132 in Medicago sativa CHS2, contains a cysteine residue (C) which plays an important catalytic role in facilitating polyketide extension beyond the triketide stage.  Another important conclusion is the observation that the S. coelicolor THNS active site does not possess an aldol switch, as is also suggested by the replacement of the highly conserved S338 by alanine .
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References

 

  • Cook, D., Rimando, A. M., Clemente, T. E., Schröder, J., Dayan, F. E., Nanayakkara, N. P. D., Pan, Z., Noonan, B. P., Fishbein, M., Abe, I., Duke, S. O., Baerson, S. R.: Alkylresorcinol synthases from Sorghum bicolor involved in the biosynthesis of the allelopathic benzoquinone sorgoleone. Plant Cell, Published on March 26, 2010; 10.1105/tpc.109.072397
    Sorghum bicolor is considered to be an allelopathic crop species, producing phytotoxins such as the lipid benzoquinone sorgoleone, which likely accounts for many of the allelopathic properties of Sorghum spp. Current evidence suggests that sorgoleone biosynthesis occurs exclusively in root hair cells and involves the production of an alkylresorcinolic intermediate (5-[(Z,Z)-8',11',14'-pentadecatrienyl]resorcinol) derived from an unusual 16:3{Delta}9,12,15 fatty acyl-CoA starter unit. This led to the suggestion of the involvement of one or more alkylresorcinol synthases (ARSs), type III polyketide synthases (PKSs) that produce 5-alkylresorcinols using medium to long-chain fatty acyl-CoA starter units via iterative condensations with malonyl-CoA. In an effort to characterize the enzymes responsible for the biosynthesis of the pentadecyl resorcinol intermediate, a previously described expressed sequence tag database prepared from isolated S. bicolor (genotype BTx623) root hairs was first mined for all PKS-like sequences. Quantitative real-time RT-PCR analyses revealed that three of these sequences were preferentially expressed in root hairs, two of which (designated ARS1 and ARS2) were found to encode ARS enzymes capable of accepting a variety of fatty acyl-CoA starter units in recombinant enzyme studies. Furthermore, RNA interference experiments directed against ARS1 and ARS2 resulted in the generation of multiple independent transformant events exhibiting dramatically reduced sorgoleone levels. Thus, both ARS1 and ARS2 are likely to participate in the biosynthesis of sorgoleone in planta. The sequences of ARS1 and ARS2 were also used to identify several rice (Oryza sativa) genes encoding ARSs, which are likely involved in the production of defense-related alkylresorcinols.
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  • Austin, M.B.; Bowman, M.E.; Ferrer, J.-L.; Schröder, J.; Noel, J.P.: An aldol switch discovered in stilbene synthases mediates cyclization specificity of type III polyketide synthases. Chemistry & Biology 11, 1179-1194 (2004).
        Stilbene synthase (STS) and chalcone synthase (CHS) each catalyze the formation of a tetraketide intermediate from a CoA-tethered phenylpropanoid starter and three molecules of malonyl-CoA, but use different cyclization mechanisms to produce distinct chemical scaffolds for a variety of plant natural products. Here we present the first STS crystal structure, and identify, by mutagenic conversion of alfalfa CHS into a functional stilbene synthase, the structural basis for the evolution of STS cyclization specificity in type III polyketide synthase (PKS) enzymes. Additional mutagenesis and enzymatic characterization confirms that electronic effects rather than steric factors balance competing cyclization specificities in CHS and STS. Finally, we discuss the problematic in vitro reconstitution of plant stilbenecarboxylate pathways, using insights from existing biomimetic polyketide cyclization studies to generate a novel mechanistic hypothesis to explain stilbenecarboxylate biosynthesis.
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