Substrate-Multiplexed Assessment of Aromatic Prenyltransferase Activity

Abstract

An increasingly effective strategy to identify synthetically useful enzymes is to sample the diversity already present in Nature. Here, we construct and assay a panel of phylogenetically diverse aromatic prenyltransferases (PTs). These enzymes catalyze a variety of C-C bond forming reactions in natural product biosynthesis and are emerging as tools for synthetic chemistry and biology. Homolog screening was further empowered through substrate-multiplexed screening, which provides direct information on enzyme specificity. We perform a head-to-head assessment of the model members of the PT family and further identify homologs with divergent sequences that rival these superb enzymes. This effort revealed the first bacterial O-Tyr PT and, together, provide valuable benchmarking for future synthetic applications of PTs.

Keywords: Biocatalysis; Enzyme Promiscuity; Multiplexed Screening; Sequence-similarity Network; Substrate Scope.

Figure 1

Substrate Multiplexed Screening of a Prenyltransferase Homolog Library. In this work, homologs were screened using a substrate mixture to assign native function, compare relative activity, and assess substrate promiscuity of the enzymes.

Figure 2

Sequence Similarity Network (SSN) of PT homologs. Selected homologs are indicated by diamonds, with colors mapped to different classes of PTs. See supporting methods for details on construction of the SSN.

Figure 3

SUMS results for PT homolog library with l‐Trp and l‐Tyr. Diamonds show the sum of all product integrations, and colored bars represent the SIR integrations for each product as a fraction of the sum of all peak integrations. Darker shades represent prenylated products, and lighter shades represent geranylated products. Bond formation at the O or C of Tyr was assigned based on product peak retention time in comparison to characterized PT homologs. Reactions consisted of whole cells resuspended in substrate mixture (1.5 mM Trp, 1.5 mM Tyr, 3 mM DMAPP, 3 mM GPP), 5 mM MgCl₂, 50 mM Tris buffer (pH=7.5), and 3 % MeOH (total volume=200 uL) at 37 °C. Reactions were quenched after 2 h. Colored boxes indicate previously uncharacterized homologs.

Figure 4

Regioselectivity of uncharacterized PT homologs. (a) Products were assigned based on overlap with products formed by known Tyr PTs. (b) For each characterized Trp PT homolog, the DMAT peak retention time was plotted against native regioselectivity to generate a box plot. (Sample size, N, at each site as follows: C4=4, C5=4, C6=6, C7=4.) For each of the four uncharacterized Trp PT homologs identified by SUMS library screening, the DMAT retention time (RT) is listed, along with the proposed regioselectivity.

Figure 5

SUMS results for PT homolog library with Trp analogs. (a) Diamonds show the sum of all product integrations, and colored bars represent the SIR integrations for each product as a fraction of the sum of all peak integrations. Only products that were detected are listed in the figure legend. Reactions consisted of whole cells resuspended in substrate mixture (0.5 mM each of iso‐Trp, 4‐Cl‐Trp, 5‐OMe‐Trp, 5‐F‐DIT, 7‐I‐Trp, and tryptamine (Tam); 3 mM each of DMAPP and GPP), 5 mM MgCl₂, 50 mM Tris buffer (pH=7.5), and 3 % MeOH (total volume=200 uL) at 37 °C. Reactions were quenched after 3 h. Gray boxes indicate homologs that were not active in the previous assay with Trp and Tyr. White boxes indicate previously uncharacterized homologs. Retention of function curves for each product are provided in Figure S5. (b) Products formed natively by select homologs from the library. Products of FscG and XptA are proposed based on biosynthetic pathway. R=prenyl.