A Promiscuous Cytochrome P450 Hydroxylates Aliphatic and Aromatic C-H Bonds of Aromatic 2,5-Diketopiperazines

Abstract

Cytochrome P450 enzymes generally functionalize inert C-H bonds, and thus, they are important biocatalysts for chemical synthesis. However, enzymes that catalyze both aliphatic and aromatic hydroxylation in the same biotransformation process have rarely been reported. A recent biochemical study demonstrated the P450 TxtC for the biosynthesis of herbicidal thaxtomins as the first example of this unique type of enzyme. Herein, the detailed characterization of substrate requirements and biocatalytic applications of TxtC are reported. The results reveal the importance of N-methylation of the thaxtomin diketopiperazine (DKP) core on enzyme reactions and demonstrate the tolerance of the enzyme to modifications on the indole and phenyl moieties of its substrates. Furthermore, hydroxylated, methylated, aromatic DKPs are synthesized through a biocatalytic route comprising TxtC and the promiscuous N-methyltransferase Amir_4628; thus providing a basis for the broad application of this unique P450.

Keywords: biocatalysis; cytochromes; enzymes; hydroxylation; structure-activity relationship.

Figure 1.

A: Select examples of multifunctional P450s indicated in this study. TxtC and FmoC are unique in catalyzing both aliphatic (in red) and aromatic (in blue) hydroxylation. Of note, TamI works along with TamL to form the ketone of tirandamycin B labelled in red. B: Schematic display of the thaxtomin biosynthetic pathway. Chemical structures of natural and unnatural thaxtomin analogues 1-6 are shown.

Figure 2.

TCB14 was the most active among four self-sufficient chimeric enzymes. A: Schematic display of four self-sufficient chimeric enzymes including linker sequences. B: The relative activities of four chimeric enzymes. The reactions contained 1.5 μm enzyme and 0.1 mm 3 and terminated after 2h. The amount of 2 in the reaction mixtures was determined at 380 nm in HPLC analysis. The amount of 2 in the TCB14 reaction was set as 100% for normalizing the relative activities of other enzymes. The data represent means ± s. d. of at least two independent experiments.

Figure 3.

Characterization of the effects of substrate N-methylations on TCB14 performance. A: HPLC analysis revealed the production of 6 in the reaction of TB14, TxtA and TxtB G554R (trace II), which missed in control carrying heat-inactivated TxtB G554R (trace I). B: HPLC analysis showed different product profiles of TCB14 reactions with 3-6 as substrates. Traces I-III represented the reactions containing 1.5 μm TCB14 and 0.1 mm substrate for 2h, while traces IV-VI showed the reactions with 15 μm TCB14 and 0.2 mm substrate for 20h. The peaks of monohydroxylated 4 and 6 were indicated with Δ, while # showed dihydroxylated 6. C: The relative activities of TCB14 toward 3-6. The amounts of monohydroxylated products in TCB14 reactions after 2h were determined at 380 nm in HPLC analysis and the highest amount (13.0 ± 0.1 μm of 2) was set as 100% for normalizing the relative activities of enzyme toward other substrates. The data represent means ± s. d. of at least two independent experiments.

Figure 4.

Biocatalytic synthesis of hydroxylated desnitro thaxtomin D analogues by TxtA, TxtB, and TCB14. A: TCB14 hydroxylated desnitro 4Me-thaxtomin D (7) that was synthesized by TxtA and TxtB (1.2 μm) after 30h (trace II) and showed the same retention time as the standard (trace III). Trace I showed the TCB14 reaction with mono- and di-hydroxylated 7 indicated by Δ and #, respectively. B: TCB14 hydroxylated 28 out of 30 desnitro thaxtomin D analogues synthesized from 5 l-Trp and 6 l-Phe analogues with conversion ratios ranging from 40.0% to 0.2%. The products were detected at 280 nm by HPLC and their concentrations were then determined by using the standard curve of cyclo-(l-Trp-l-Phe). The conversion ratios of mono- and dihydroxylated products were shown as solid and sliced bars, respectively. The data represent means of at least two independent experiments with details shown in Table S2.

Figure 5.

Biocatalytic synthesis of hydroxylated, methylated aromatic DKPs using TCB14 and Amir_4628. A: HPLC analysis revealed the formation of methylated cWFs and hydroxylated cWF-Me₂. Trace I represented the negative control containing heat-inactivated Amir_4628, while traces II and III showed the full reactions with SAM or hMAT2A that generated SAM in situ, respectively. Trace IV indicated the above reaction after adding TCB14. The peaks of mono- and di-hydroxylated cWF-Me₂ were indicated with Δ and #, respectively. B: Amir_4628 methylated six aromatic DKPs to a varying extent. Mono- and dimethylated products were shown as orange and green bars, respectively. The complete consumption of DKP substrates was set as 100% of the conversion ratio. The data represent means ± s. d. of at least two independent experiments. C: HPLC analysis revealed that TCB14 was able to produce monohydroxylated cWW-Me₂, cWY-Me₂, and cLF-Me₂ as indicated by Δ.