Evolution of aromatic prenyltransferases in the biosynthesis of indole derivatives

SHU-MING LI
Institut für Pharmazeutische Biologie und Biotechnologie
der Universität Düsseldorf
Universitätsstraße 1, Geb. 26.23
D-40225 Düsseldorf
shuming.li@uni-duesseldorf.de
http://www.rz.uni-duesseldorf.de/WWW/MathNat/PharmBio/

Literature:
1. Anika Kremer, Lucia Westrich and Shu-Ming Li (2007). A 7-dimethylallyltryptophan synthase from Aspergillus fumigatus: Overproduction, purification and biochemical characterization. Microbiology, in press.
A putative prenyltransferase gene, Afu3g12930, was identified in the genome sequence of Aspergillus fumigatus. EAL92290, encoded by Afu3g12930, consists of 472 amino acids with a molecular weight of about 53 kDa. The coding sequence of Afu3g12930 was cloned in pQE60 and overexpressed in Escherichia coli. The soluble His₆-fusion protein was purified to apparent homogeneity and characterized biochemically. The enzyme was found to catalyse the prenylation of tryptophan at C7 position of the indole moiety in the presence of dimethylallyl diphosphate (DMAPP) and therefore, functions as a 7-dimethylallyltryptophan synthase (7-DMATS). The structure of the enzymic product was elucidated by NMR and MS analysis. K_m values were determined for DMAPP at 67 µM and for L-tryptophan at 137 µM, respectively. Geranyl diphosphate was not accepted as prenyl donor, while tryptophan-containing dipeptides were also aromatic substrates of 7-DMATS. 7-DMATS did not need divalent metal ions for its enzymic reaction, although Ca²⁺ enhanced the reaction velocity slightly.
7-DMATS is the second dimethylallyltryptophan synthase identified in Aspergillus fumigatus. Interestingly, it shares only a sequence identity of 31 % on the amino acid level with another known dimethylallyltryptophan synthase FgaPT2 from the same fungus, which prenylates L-tryptophan at C4 position of the indole ring.
Afu3g12930 belongs to a putative biosynthetic gene cluster consisting of eight genes. Orthologous clusters were also identified in the genome sequences of Neosartorya fischeri and Aspergillus terreus. The putative roles of the genes in the cluster are discussed.

2. Nicola Steffan, Inge A. Unsöld and Shu-Ming Li (2007). Chemoenzymatic synthesis of indole derivatives using a dimethylallyltryptophan synthases from Aspergillus fumigatus. ChemBiochem, DOI: 10.1002/cbic.200700107
A 4-dimethylallyltryptophan synthase, FgaPT2, has been identified in the genome of Aspergillus fumigatus. In a previous study, FgaPT2 was overexpressed in Saccharomyces cerevisiae and characterized biochemically. A higher protein yield (up to 100-fold higher than that for S. cerevisiae) has now been achieved by overexpression in E. coli; this has permitted investigation into substrate specificity with alternative substances. FgaPT2 accepted 17 of 37 commercially available indole derivatives as substrates. Tryptophan derivatives that carry methyl groups at the indole ring showed a different acceptance from those with methyl groups on the side chain. 5-Hydroxytryptophan was well accepted by FgaPT2, while the halogenated derivatives were not accepted. Decarboxylation, deamination, or oxidative deamination of tryptophan, as well as replacement of the NH2 group by OH, or of the COOH group by CH₂COOH or CONHOH resulted in decreased but still significant enzymatic activity. None of the tested tryptophan-containing dipeptides was accepted by FgaPT2. Structural elucidation of isolated enzymatic products by NMR and MS analyses proved unequivocally that the prenylation was regioselective at position C4 of the indole ring in the presence of dimethylallyl diphosphate. Determination of the kinetic parameters revealed that L-tryptophan was accepted as the best substrate by the enzyme, followed by 5-,6-,7-methyltryptophan and L-abrine. The enzymatic rate constant (k_cat K_m^-1) of nine selected substrates were found to be about 1.0 to 6.5 % of that for L-tryptophan. Overnight incubation with eight substances showed that the conversion ratio to their prenylated derivatives was in the range 32.5 to 99.7 %. This provides evidence that 4-dimethylallylated indole derivatives can be produced by chemoenzymatic synthesis with FgaPT2.

3. Wen-Bing Yin, Han-Li Ruan, Lucia Westrich, Alexander Grundmann and Shu-Ming Li (2007). CdpNPT, a N-prenyltransferase from Aspergillus fumigatus: Overproduction, purification and biochemical characterization. ChemBiochem, DOI: 10.1002/cbic.200700079
A putative prenyltransferase gene, cdpNPT, was identified in the genome sequence of Aspergillus fumigatus by a homology search by using known prenyltransferases and sequence analysis. CdpNPT consists of 440 amino acids and has a molecular mass of about 50 kDa. The coding sequence of cdpNPT was cloned in pQE60 and overexpressed in E. coli. The soluble His₆-fusion CdpNPT was purified to near homogeneity and characterised biochemically. The enzyme showed broad substrate specificity towards aromatic substrates and was found to catalyse the prenylation of tryptophan-containing cyclic dipeptides at N1 of the indole moieties in the presence of dimethylallyl diphosphate (DMAPP); geranyl diphosphate was not accepted as prenyl donor. The structures of the enzymatic products were elucidated by NMR and MS analysis. The K_m value for DMAPP was determined to be 650 µM. Due to substrate inhibition, K_m values could not be obtained for the aromatic substrates. CdpNPT does not need divalent metal ions for its enzymatic reaction, although Ca²⁺ enhances the reaction velocity by up to the threefold. CdpNPT is the first N-prenyltransferase that has been purified and characterised in a homogenous form after heterologous overproduction. Interestingly, it shows significant sequence similarity to other indole prenyltransferases that catalyse the formation of C-C bonds.

4. Shu-Ming Li and Inge A. Unsöld (2006). Post genome research in the biosynthesis of ergot alkaloids. Planta Medica 72:1117-1120.
Genome sequencing provides new opportunities and challenges to identify genes for the biosynthesis of secondary metabolites. A putative biosynthetic gene cluster of fumigaclavine C, an ergot alkaloid of the clavine type, was identified in the genome sequence of Aspergillus fumigatus by a bioinformatic approach. This cluster spans 22 kb of genomic DNA and comprises at least 11 open reading frames (ORFs). Seven of them are orthologous to genes from the biosynthetic gene cluster of ergot alkaloids in Claviceps purpurea. Experimental evidence of the identified cluster was provided by heterologous expression and biochemical characterization of two ORFs, FgaPT1 and FgaPT2, in the cluster of A. fumigatus, which show remarkable similarities to dimethylallyltryptophan synthase from C. purpurea and function as prenyltransferases. FgaPT2 converts L-tryptophan to dimethylallyltryptophan and thereby catalyzes the first step of ergot alkaloid biosynthesis, whilst FgaPT1 catalyzes the last step of the fumigaclavine C biosynthesis, i.e. the prenylation of fumigaclavine A at C-2 position of the indole nucleus. In addition to information obtained from the gene cluster of ergot alkaloids from C. purpurea, the identification of the biosynthetic gene cluster of fumigaclavine C in A. fumigatus opens an alternative way to study the biosynthesis of ergot alkaloids in fungi.

5. Inge A. Unsöld and Shu-Ming Li (2006). Reverse prenyltransferase in the biosynthesis of fumigaclavine c in Aspergillus fumigatus: gene expression, purification and characterization of fumigaclavine C synthase FgaPT1. ChemBioChem 7:158-164.
A putative prenyltransferase gene, fgaPT1, was identified in the biosynthetic gene cluster of fumigaclavines in Aspergillus fumigatus AF293. fgaPT1 was cloned and overexpressed in Escherichia coli. The His₆-fusion FgaPT1 was purified to near homogeneity and characterized biochemically. The enzyme was found to convert fumigaclavine A to fumigaclavine C by attaching a dimethylallyl moiety to C-2 of the indole nucleus in a "reverse" manner, i.e. by connection of C-3 of the dimethylallyl moiety to an aromatic nucleus. FgaPT1 is a soluble, dimeric protein with a subunit size of 50 kDa. K_m(app) values for fumigaclavine A and dimethylallyl diphosphate were determined as 6 and 13 µM, respectively. The turnover number was 0.8 s^-1. Metal ions such as Mg²⁺ and Ca²⁺ are not essential for the enzymatic activity. FgaPT1 showed relatively strict substrate specificity towards fumigaclavine A. Only dimethylallyl diphosphate was accepted as prenyl diphosphate under our conditions.
FgaPT1 is the first reverse prenyltransferase from fungi which has been purified and characterized in homogenous form after heterologous overproduction. Surprisingly, it shows very low sequence similarity to the recently identified prenyltransferase LtxC from cyanobacteria, which also catalyzes the reverse prenylation of an indole nucleus.

6. Alexander Grundmann and Shu-Ming Li (2005). Overproduction, purification and characterization of FtmPT1, a brevianamide F prenyltransferase from Aspergillus fumigatus. Microbiology 151: 2199-2207.
A putative prenyltransferase gene, ftmPT1, was identified in the genome sequence of Aspergillus fumigatus. ftmPT1 was cloned and expressed in Escherichia coli, and the protein FtmPT1 was purified to near homogeneity and characterized biochemically. This enzyme was found to catalyse the prenylation of cyclo-L-trp-L-Pro (brevianamide F) at the C-2 position of the indole nucleus. FtmPT1 is a soluble monomeric protein, which does not contain the usual prenyl diphosphate binding site (N/D)DXXD found in most prenyltransferases, and which does not require divalent metal ions for its enzymic activity. K_m values for brevianamide F and dimethylallyl diphosphate were determined as 55 and 74 µM, respectively. The turnover number was 5·57 s^-1. FtmPT1 showed a high substrate specificity towards dimethylallyl diphosphate, but accepted different tryptophan-containing cyclic dipeptides. Together with dimethylallyltryptophan synthase of ergot alkaloid biosynthesis, FtmPT1 belongs to a new group of prenyltransferases with aromatic substrates.

7. Inge A. Unsöld und Shu-Ming Li (2005). Overproduction, purification and characterization of FgaPT2, a dimethylallyltryptophan synthase from Aspergillus fumigatus, Microbiology 151: 1499-1505.
A putative dimethylallyltryptophan synthase gene, fgaPT2, was identified in the genome sequence of Aspergillus fumigatus. fgaPT2 was cloned and overexpressed in Saccharomyces cerevisiae. The protein FgaPT2 was purified to near homogeneity and characterized biochemically. This enzyme was found to convert L-tryptophan to 4-dimethylallyltryptophan, a reaction known to be the first step in ergot alkaloid biosynthesis. FgaPT2 is a soluble, dimeric protein with a subunit size of 52 kDa, and contains no putative prenyl diphosphate binding site (N/D)DXXD. K_m values for L-tryptophan and dimethylallyl diphosphate (DMAPP) were determined as 8 and 4 µM, respectively. Metal ions, such as Mg²⁺ and Ca²⁺, enhance the reaction velocity, but are not essential for the enzymic reaction. FgaPT2 showed a relatively strict substrate specificity for both tryptophan and DMAPP. FgaPT2 is the first enzyme in the biosynthesis of ergot alkaloids to be purified and characterized in homogeneous form after heterologous overproduction.

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