Targeting active site residues and structural anchoring positions in terpene synthases

The sesterterpene synthase SmTS1 from Streptomyces mobaraensis contains several unusual residues in positions that are otherwise highly conserved. Site-directed mutagenesis experiments for these residues are reported that showed different effects, resulting in some cases in an improved catalytic activity, but in other cases in a loss of enzyme function. For other enzyme variants a functional switch was observed, turning SmTS1 from a sesterterpene into a diterpene synthase. This article gives rational explanations for these findings that may generally allow for protein engineering of other terpene synthases to improve their catalytic efficiency or to change their functions.

. Amino acid sequence alignment some characterised sesqui-and diterpene synthases. Highly conserved residues and motifs are marked in yellow.

Site-directed mutagenesis
The site mutations were performed by the overlap extension PCR (OE-PCR) method. [1] The template was the wildtype gene cloned into the pYE-Express expression vector. [2] The mutational primers are listed in Table S1, the polymerase was purchased from NEB (Q5® High-Fidelity DNA polymerase, Ipswich, Massachusetts, USA). The first-round PCRs for individual amplification of the left and the right part of the mutated gene followed the program: 1) 98 °C for 30 s; 2) 98 °C for 10 s, 67 °C for 30 s, 72 °C for 40 s; repeated 32 times; 3) 72 °C for 2 min. The PCR products were analyzed by gel electrophoresis and purified by the Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, Wisconsin, USA). The obtained overlapping fragments were then mixed and were used for the next round PCRs over two steps. The mutated genes were cloned into the pYE-Express expression vector by homologous recombination in yeast following the standard PEG/LiOAc/salmon sperm protocol. [3,4]

Expression and purification of SmTS1 variants
The transformants containing the mutations were inoculated in LB broth (3 mL, kanamycin
Production of compounds 1 -6 by peak integration of total ion chromatograms from triplicates. Production of 1 and co-eluting 6 by wildtype SmTS1 is set to 100%.
[b] Enzyme activities were calculated from total production of all compounds by peak integrations from triplicates. Activity of wildtype SmTS1 is set to 100%. S11 Figure S4. Production of compounds 1-6 and a-c by peak integration of total ion chromatograms (mean ± standard deviation from triplicates). Production of 1 with coeluting 6 by wildtype SmTS1 is set to 100%. The production of A) 2, B) 1 + 6, C) unknown a, D) unknown b + c, E) 3 + 4, F) 5 and G) total activities of the wildtype and each variant. For a comparison of enzyme activities, the peak integrals of all produced compounds were summarised. Activity of wildtype SmTS1 is set to 100%. S12 Figure S5. The A222V enzyme variant. A) Total ion chromatogram of an extract from an incubation with GGPP, and mass spectra of B) 8 and C) 9. S13 Table S3. Production of sesterterpens and and diterpenes by wildtype SmTS1 and its enzyme variants related to the position A222, and relative enzyme activities.  100%. [b] Enzyme activities were calculated from total production of 8 and 9 by peak integrations from triplicates. S14 Figure S6. Total ion chromatograms of extracts from incubations of GFPP with A) wildtype SmTS1, and the SmTS1 variants B) A222M, and C) A222L. S15 Figure S7. Total ion chromatograms of extracts from incubations of GGPP with the SmTS1 variants A) A222V, B) A222M, C) A222I, D) A222L, and E) A222F. Asterisks indicate degradation products from GGPP also observed without enzyme. S16 Figure S8. Production of sesterterpenes and diterpenes from SmTS1 and its variants in the position of A222. Production of A) sesterterpenes, B) compound 8, C) compound 9 and D) diterpenes (sum of 8 and 9) by the wildtype and its variants. The data were obtained by peak integration of total ion chromatograms (mean ± standard deviation from triplicates). Production of sesterterpenes by wildtype is set to 100%. S17 Figure S9. Structure elucidation of 8. Bold: 1 H, 1 H COSY, single headed arrows: key HMBC, and double headed arrows: key NOESY correlations. Carbon numbering follows GGPP numbering to indicate the origin of each carbon.  Figure S17. Structure elucidation of 9. Bold: 1 H, 1 H-COSY, single headed arrows: key HMBC, and double headed arrows: key NOESY correlations. Carbon numbering follows GGPP numbering to indicate the origin of each carbon.