# Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids
## 2 Dityptophanine  
Ditryptophenaline **(1)** is a dimeric diketopiperazine alkaloid [5] and has a complicated structure (Figure 2). Dimerization is found relatively frequently among natural products, as it is considered to be an efficient way of enhancing the chemical complexity and diversity of metabolites being biosynthesized. It can also increase the valency of a compound, affording the molecule a higher affinity for its target or higher stability upon forming a complex with its target [6]. Compound **1**[7] and its related compound WIN 64821 (Figure 2) [8] originally isolated from _Aspergillus versicolor_ exhibit useful biological activities, such as an inhibition of substance P receptor for potential analgesic and anti-inflammatory activities. Despite their complex chemical structures, their formation seemed relatively simple, involving dimerization of diketopiperazine monomers formed through condensation of two amino acids at the initial stage. Judging from the structure of the diketopiperazine monomer of **1**, it appeared that L-tryptophan and L-phenylalanine could be condensed together to form the diketopiperazine core structure of the monomer. Once the phenylalanine-derived amide nitrogen of the diketopiperazine core is \(N\)-methylated, a ring closure within the monomer and a subsequent monomer-monomer crosslinking through oxidation reactions involving the tryptophan indole ring can lead to the formation of a homodimer of **1**. Figure 1: Chemical structures of alkaloids in a broad sense: natural alkaloids of non-plant origin, tetrodotoxin and saxitoxin; a natural but non-basic alkaloid, colchicine; and a synthetic alkaloid, procaine. Figure 2: Chemical structures of dimeric diketopiperazine alkaloids, (–)-ditryptophenaline **1**[7] and (+)-WIN 64821 [8]. More recently, **1** was found to be produced by another fungus, _Aspergillus flavus_[9]. To elucidate the biosynthetic pathway of **1**, experiments of deficiency of the biosynthetic gene were performed to identify ditryptophenaline biosynthetic gene cluster. Based on the homology of amino acid sequences deduced from the _A. flavus_ genome sequence [10] to known enzymes, three genes, _dtpA_, _dtpB_ and _dtpC_, clustered together on the _A. flavus_ genome were predicted to code for a nonribosomal peptide synthetase (NRPS), an \(N\)-methyltransferasease and a cytochrome P450 (P450), respectively (Figure 3) [9]. Knockout of any one of the three genes in the _A. flavus_\(\Delta\)_pyrG_/\(\Delta\)_ku70_ strain A1421 [11] resulted in the loss of production of **1**, indicating that these three genes were involved in the biosynthesis of **1**. Based on the in vitro assays of DtpB using a substrate **2** isolated from the _dtpB_ knockout mutant, DtpB was shown to catalyze an \(N\)-methylation of **2** to biosynthesize the intermediate **3**. Interestingly, **3** was directly converted to **1** by DtpC (P450), which was prepared as a microsomal fraction of _Saccharomyces cerevisiae_ expressing _dtpC_ recombinantly. The analysis of the in vitro experiments suggested that DtpC catalyzes the C2-N10 ring closure within the monomer and the dimerization of the tetracycline monomers, both of which likely proceed through a radical pathway. In the proposed mechanism (Figure 4), a radical formed at N10 through hydrogen atom abstraction by the heme iron of P450 can migrate to C3 with a concomitant C2-N10 ring closure and undergo a radical-mediated coupling with another C3 radical-bearing monomer to form the bridging bond in the dimer like **1**. Figure 4: Proposed mechanism of DtpC-catalyzed dimerization of intermediates **3** and **4** to form (\(-\))-ditryptophenaline **1**, (\(-\))-dibrevianamide F **5** and (\(-\))-trypophenaline **6** via a radical route [9]. DtpA, ditryptophenaline nonribosomal peptide synthetase (NRPS); DtpB, \(N\)-methyltransferasease; DtpC, cytochrome P450; FtmA, füntremorgin NRPS. Figure 3: _Aspergillus flavus_ ditryptophenaline biosynthetic gene cluster and predicted functions of the genes in the cluster.