Dae-Yeon Suh

LAP6/POLYKETIDE SYNTHASE A and LAP5/POLYKETIDE SYNTHASE B Encode Hydroxyalkyl α-Pyrone Synthases Required for Pollen Development and Sporopollenin Biosynthesis in Arabidopsis thaliana

This article characterizes two anther-expressed type III polyketide synthases that are related to chalcone synthase. The results support the hypothesis that the enzymes are involved in an ancient sporopollenin biosynthetic pathway that catalyzes sequential modification of fatty acid starter molecules to generate alkyl α-pyrone polyketide sporopollenin components of the pollen exine. Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide α-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain- and ω-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated α-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors.

Divergent evolution of the thiolase superfamily and chalcone synthase family

Enzymes of the thiolase superfamily catalyze the formation of carbon-carbon bond via the Claisen condensation reaction. Thiolases catalyze the reversible non-decarboxylative condensation of acetoacetyl-CoA from two molecules of acetyl-CoA, and possess a conserved Cys-His catalytic diad. Elongation enzymes (beta-ketoacyl-acyl carrier protein synthase (KAS) I and KAS II and the condensing domain of polyketide synthase) have invariant Cys and two His residues (CHH triad), while a Cys-His-Asn (CHN) triad is found in initiation enzymes (KAS III, 3-ketoacyl-CoA synthase (KCS) and the chalcone synthase (CHS) family). These enzymes all catalyze decarboxylative condensation reactions. 3-Hydroxyl-3-methylglutaryl-CoA synthase (HMGS) also contains the CHN triad, although it catalyzes a non-decarboxylative condensation. That the enzymes of the thiolase superfamily share overall similarity in protein structure and function suggested a common evolutionary origin. All thiolases were found to have, in addition to the Cys-His diad, either Asn or His (thus C(N/H)H) at a position corresponding to the His in the CHH and CHN triads. In our phylogenetic analyses, the thiolase superfamily was divided into four main clusters according to active site architecture. During the functional divergence of the superfamily, the active architecture was suggested to evolve from the C(H)H in archaeal thiolases to the C(N/H)H in non-archaeal thiolases, and subsequently to the CHH in the elongation enzymes and the CHN in the initiation enzymes. Based on these observations and available biochemical and structural evidences, a plausible evolutionary history for the thiolase superfamily is proposed that includes the emergence of decarboxylative condensing enzymes accompanied by a recruitment of the His in the CHH and CHN triads for a catalytic role during decarboxylative condensation. In addition, phylogenetic analysis of the plant CHS family showed separate clustering of CHS and non-CHS members of the family with a few exceptions, suggesting repeated gene birth-and-death and re-invention of non-CHS functions throughout the evolution of angiosperms. Based on these observations, predictions on the enzymatic functions are made for several members of the CHS family whose functions are yet to be characterized. Further, a moss CHS-like enzyme that is functionally similar to a cyanobacterial enzyme was identified as the most recent common ancestor to the plant CHS family.

Cross-reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli

Chalcone synthase (CHS) and stilbene synthase (STS) are related plant polyketide synthases belonging to the CHS superfamily. CHS and STS catalyze common condensation reactions of p‐coumaroyl‐CoA and three C2‐units from malonyl‐CoA but different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Using purified Pueraria lobata CHS and Arachis hypogaea STS overexpressed in Escherichia coli, bisnoryangonin (BNY, the derailed lactone after two condensations) and p‐coumaroyltriacetic acid lactone (the derailed lactone after three condensations) were detected from the reaction products. More importantly, we found a cross‐reaction between CHS and STS, i.e. resveratrol production by CHS (2.7–4.2% of naringenin) and naringenin production by STS (1.4–2.3% of resveratrol), possibly due to the conformational flexibility of their active sites.

Genome-wide analysis of the chalcone synthase superfamily genes of Physcomitrella patens

Enzymes of the chalcone synthase (CHS) superfamily catalyze the production of a variety of secondary metabolites in bacteria, fungi and plants. Some of these metabolites have played important roles during the early evolution of land plants by providing protection from various environmental assaults including UV irradiation. The genome of the moss, Physcomitrella patens, contains at least 17 putative CHS superfamily genes. Three of these genes (PpCHS2b, PpCHS3 and PpCHS5) exist in multiple copies and all have corresponding ESTs. PpCHS11 and probably also PpCHS9 encode non-CHS enzymes, while PpCHS10 appears to be an ortholog of plant genes encoding anther-specific CHS-like enzymes. It was inferred from the genomic locations of genes comprising it that the moss CHS superfamily expanded through tandem and segmental duplication events. Inferred exon–intron architectures and results from phylogenetic analysis of representative CHS superfamily genes of P. patens and other plants showed that intron gain and loss occurred several times during evolution of this gene superfamily. A high proportion of P. patensCHS genes (7 of 14 genes for which the full sequence is known and probably 3 additional genes) are intronless, prompting speculation that CHS gene duplication via retrotransposition has occurred at least twice in the moss lineage. Analyses of sequence similarities, catalytic motifs and EST data indicated that a surprisingly large number (as many as 13) of the moss CHS superfamily genes probably encode active CHS. EST distribution data and different light responsiveness observed with selected genes provide evidence for their differential regulation. Observed diversity within the moss CHS superfamily and amenability to gene manipulation make Physcomitrella a highly suitable model system for studying expansion and functional diversification of the plant CHS superfamily of genes.

Crystal structure of stilbene synthase from Arachis hypogaea

Introduction. Stilbene synthase [STS; Enzyme Commission (EC) 2.3.1.95] and chalcone synthase (CHS; EC 2.3.1.74) are members of the type III polyketide synthases (PKSs) and plant-specific enzymes. 1 CHS is widely found in higher plants and plays a key role in the flavonoid biosynthesis by supplying chalcone to downstream enzymes. In contrast, a limited number of plants have STS essential for the synthesis of resveratrol utilized in the stilbenoid biosynthesis. 2 The members of CHS superfamily, including STS, produce linear polyketide intermediates by a common catalytic mechanism where coenzyme A (CoA)-linked starter molecules are iteratively condensed by acetyl units from malonyl-CoA. 3 STS and CHS share around 70% sequence identity without significant deletions and insertions; therefore, the enzymatic mechanism of STS has been considered to be very close to that of CHS. STS and CHS catalyze condensation reactions of pcoumaroyl-CoA and 3 acetyl units from malonyl-CoA, and produce a common linear tetraketide intermediate. In the following cyclization reaction, however, STS and CHS catalyze aldol and Claisen condensation of the tetraketide, resulting in 2 different final products, resveratrol and chalcone, respectively. The crystal structure and molecular mechanism of CHS from Medicago sativa (alfalfa) have recently been reported, 4 but the primary determinant of the cyclization reactions catalyzed by STS and CHS was not clear. More recently, the crystal structure of STS from Pinus silvestris (pine) was reported and provided a framework for understanding the specificity in the cyclization reaction. 5 This report describes the crystal structures of STS from Arachis hypogaea (peanut) in the absence and presence of its final product resveratrol at 2.4 A and 2.9 A, respectively. Detailed structural comparisons of STS from A. hypogaea (peanut STS) with STS from P. silvestris (pine STS) and CHS (alfalfa CHS) evidently revealed common differences between STS and CHS in the local conformation around the active site pocket.

PpASCL, a moss ortholog of anther-specific chalcone synthase-like enzymes, is a hydroxyalkylpyrone synthase involved in an evolutionarily conserved sporopollenin biosynthesis pathway

Sporopollenin is the main constituent of the exine layer of spore and pollen walls. Recently, several Arabidopsis genes, including polyketide synthase A (PKSA), which encodes an anther-specific chalcone synthase-like enzyme (ASCL), have been shown to be involved in sporopollenin biosynthesis. The genome of the moss Physcomitrella patens contains putative orthologs of the Arabidopsis sporopollenin biosynthesis genes. We analyzed available P.patens expressed sequence tag (EST) data for putative moss orthologs of the Arabidopsis genes of sporopollenin biosynthesis and studied the enzymatic properties and reaction mechanism of recombinant PpASCL, the P.patens ortholog of Arabidopsis PKSA. We also generated structure models of PpASCL and Arabidopsis PKSA to study their substrate specificity. Physcomitrella patens orthologs of Arabidopsis genes for sporopollenin biosynthesis were found to be expressed in the sporophyte generation. Similarly to Arabidopsis PKSA, PpASCL condenses hydroxy fatty acyl-CoA esters with malonyl-CoA and produces hydroxyalkyl α-pyrones that probably serve as building blocks of sporopollenin. The ASCL-specific set of Gly-Gly-Ala residues predicted by the models to be located at the floor of the putative active site is proposed to serve as the opening of an acyl-binding tunnel in ASCL. These results suggest that ASCL functions together with other sporophyte-specific enzymes to provide polyhydroxylated precursors of sporopollenin in a pathway common to land plants.

PpASCL, the Physcomitrella patens Anther-Specific Chalcone Synthase-Like Enzyme Implicated in Sporopollenin Biosynthesis, Is Needed for Integrity of the Moss Spore Wall and Spore Viability.

Sporopollenin is the main constituent of the exine layer of spore and pollen walls. The anther-specific chalcone synthase-like (ASCL) enzyme of Physcomitrella patens, PpASCL, has previously been implicated in the biosynthesis of sporopollenin, the main constituent of exine and perine, the two outermost layers of the moss spore cell wall. We made targeted knockouts of the corresponding gene, PpASCL, and phenotypically characterized ascl sporophytes and spores at different developmental stages. Ascl plants developed normally until late in sporophytic development, when the spores produced were structurally aberrant and inviable. The development of the ascl spore cell wall appeared to be arrested early in microspore development, resulting in small, collapsed spores with altered surface morphology. The typical stratification of the spore cell wall was absent with only an abnormal perine recognisable above an amorphous layer possibly representing remnants of compromised intine and/or exine. Equivalent resistance of the spore walls of ascl mutants and the control strain to acetolysis suggests the presence of chemically inert, defective sporopollenin in the mutants. Anatomical abnormalities of late-stage ascl sporophytes include a persistent large columella and an air space incompletely filled with spores. Our results indicate that the evolutionarily conserved PpASCL gene is needed for proper construction of the spore wall and for normal maturation and viability of moss spores.

Hypericum perforatum hydroxyalkylpyrone synthase involved in sporopollenin biosynthesis – phylogeny, site‐directed mutagenesis, and expression in nonanther tissues

Anther‐specific chalcone synthase‐like enzyme (ASCL), an ancient plant type III polyketide synthase, is involved in the biosynthesis of sporopollenin, the stable biopolymer found in the exine layer of the wall of a spore or pollen grain. The gene encoding polyketide synthase 1 from Hypericum perforatum (HpPKS1) was previously shown to be expressed mainly in young flower buds, but also in leaves and other tissues at lower levels. Angiosperm ASCLs, identified by sequence and phylogenetic analyses, are divided into two sister clades, the Ala‐clade and the Val‐clade, and HpPKS1 belongs to the Ala‐clade. Recombinant HpPKS1 produced triketide and, to a lesser extent, tetraketide alkylpyrones from medium‐chain (C6) to very long‐chain (C24) fatty acyl‐CoA substrates. Like other ASCLs, HpPKS1 also preferred hydroxyl fatty acyl‐CoA esters over the analogous unsubstituted fatty acyl‐CoA esters. To study the structural basis of the substrate preference, mutants of Ala200 and Ala215 at the putative active site and Arg202 and Asp211 at the modeled acyl‐binding tunnel were constructed. The A200T/A215Q mutant accepted decanoyl‐CoA, a poor substrate for the wild‐type enzyme, possibly because of active site constriction by bulkier substitutions. The substrate preference of the A215V and A200T/A215Q mutants shifted toward nonhydroxylated, medium‐chain to long‐chain fatty acyl‐CoA substrates. The R202L/D211V double mutant was selective for acyl‐CoA with chain lengths of C16–C18, and showed a diminished preference for the hydroxylated acyl‐CoA substrates. Transient upregulation by abscisic acid and downregulation by jasmonic acid and wounding suggested that HpPKS1, and possibly other Ala‐clade ASCLs, may be involved in the biosynthesis of minor cell wall components in nonanther tissues.

Physcomitrella PpORS, Basal to Plant Type III Polyketide Synthases in Phylogenetic Trees, Is a Very Long Chain 2′-Oxoalkylresorcinol Synthase

Background: Physcomitrella PpORS is an ancient member of the plant type III polyketide synthase (PKS) family. Results: PpORS, produced in nonprotonemal moss cells, synthesizes pentaketide 2′-oxoalkylresorcinols using a unique substrate binding site. Conclusion: PpORS is a novel very long chain 2′-oxoalkylresorcinol synthase. Significance: This is the first step toward understanding the co-evolution of the type III PKS family and land plants. The plant type III polyketide synthases (PKSs), which produce diverse secondary metabolites with different biological activities, have successfully co-evolved with land plants. To gain insight into the roles that ancestral type III PKSs played during the early evolution of land plants, we cloned and characterized PpORS from the moss Physcomitrella. PpORS has been proposed to closely resemble the most recent common ancestor of the plant type III PKSs. PpORS condenses a very long chain fatty acyl-CoA with four molecules of malonyl-CoA and catalyzes decarboxylative aldol cyclization to yield the pentaketide 2′-oxoalkylresorcinol. Therefore, PpORS is a 2′-oxoalkylresorcinol synthase. Structure modeling and sequence alignments identified a unique set of amino acid residues (Gln218, Val277, and Ala286) at the putative PpORS active site. Substitution of the Ala286 to Phe apparently constricted the active site cavity, and the A286F mutant instead produced triketide alkylpyrones from fatty acyl-CoA substrates with shorter chain lengths. Phylogenetic analysis and comparison of the active sites of PpORS and alkylresorcinol synthases from sorghum and rice suggested that the gramineous enzymes evolved independently from PpORS to have similar functions but with distinct active site architecture. Microarray analysis revealed that PpORS is exclusively expressed in nonprotonemal moss cells. The in planta function of PpORS, therefore, is probably related to a nonprotonemal structure, such as the cuticle.

Mapping the Mechanism of the Resorcinol Ring Formation Catalyzed by ArsB, a Type III Polyketide Synthase from Azotobacter vinelandii

Whos first? Aldol cyclization occurs before hydrolysis in the resorcinol ring formation catalyzed by the type III polyketide synthase, ArsB. Synthetic C(20)-TKA was not converted to alkylresorcinol by ArsB, but rather inhibited the enzyme activity, thus indicating that C(20)-TKA is not an intermediate in ArsB-catalyzed alkylresorcinol formation.