Expression of presumptive Microbial terpene synthase like genes (MTPSLs) from Lophocolea bidentata (liverwort) in moss Physcomitrella patens
Author
Kodiripaka, Satish Kumar
Term
4. term
Education
Publication year
2020
Submitted on
2020-06-03
Pages
67
Abstract
Terpenoider er en stor og mangfoldig gruppe naturstoffer, som er meget udbredt i planter og sjældnere i mikroorganismer. De dannes af enzymer kaldet terpensyntaser, der omdanner prenyl-difosfat-forløbere til mange forskellige terpener. Gener, der ligner mikrobielle terpensyntaser (MTPSL), er især fundet i ikke-frøplanter, men ikke i frøplanter. I dette arbejde fokuserede vi på fire sådanne gener (LbMTPSL1, 3, 4 og 5) fra levermosen Lophocolea bidentata. Målet var at udtrykke og biokemisk karakterisere dem i modelplanten Physcomitrella patens, som udnytter effektiv homolog rekombination. For at understøtte dette etablerede vi en lovende protokol til fremstilling af protoplaster (planteceller uden cellevæg) fra P. patens ved at kombinere enzymerne Cellulase R-10 og Macerozyme R-10. Vi byggede desuden homologi-baserede strukturmodeller. LbMTPSL1 og LbMTPSL4 havde størst strukturel lighed med epi-isozizaen-syntase fra Streptomyces coelicolor, mens LbMTPSL3 og LbMTPSL5 lignede selinadien-syntase fra Streptomyces pristinaespiralis; begge tilhører C15-underklassen (sesquiterpensyntaser). Strukturbaserede funktionsanalyser forudsagde, at det aktive center binder farnesyl-difosfat (FPP) for LbMTPSL1, 3 og 5, og den beslægtede analog farnesyl thiopyrophosphate (FsPP) for LbMTPSL4. I alle fire enzymer identificerede vi tre basiske aminosyrer sammen med en fenolholdig aminosyre i et substratgenkendelsesmotiv samt tre aromatiske aminosyrer, der via kation–π-interaktioner kan stabilisere karbokation-mellemliggende. Disse beregningsbaserede indsigter i LbMTPSL-proteinerne giver et vigtigt udgangspunkt for fremtidige mekanistiske studier og rationelt design, der kan manipulere FPP-cykliseringsbaner og dermed ændre produktprofilen betydeligt.
Terpenoids are a large and diverse family of natural compounds, abundant in plants and rarer in microbes. They are made by enzymes called terpene synthases, which convert prenyl diphosphate precursors into many different terpenes. Genes that resemble microbial terpene synthases (MTPSLs) have been identified mainly in non-seed plants, but not in seed plants. Here, we focused on four such genes (LbMTPSL1, 3, 4, and 5) from the liverwort Lophocolea bidentata. Our aim was to heterologously express and biochemically characterize them in the model plant Physcomitrella patens, which supports efficient homologous recombination. To enable this, we established a promising protoplast preparation protocol for P. patens using a blend of Cellulase R-10 and Macerozyme R-10; protoplasts are plant cells without their cell wall that facilitate DNA uptake. We also built homology-based structural models. LbMTPSL1 and LbMTPSL4 showed the closest similarity to epi-isozizaene synthase from Streptomyces coelicolor, while LbMTPSL3 and LbMTPSL5 resembled selinadiene synthase from Streptomyces pristinaespiralis; both are C15 (sesquiterpene) synthases. Structure-based analyses predicted active-site binding to farnesyl diphosphate (FPP) for LbMTPSL1, 3, and 5, and to the analogue farnesyl thiopyrophosphate (FsPP) for LbMTPSL4. Across all four enzymes, we identified three basic residues together with a phenol-containing amino acid in a substrate recognition motif, and three aromatic residues that could stabilize carbocation intermediates via cation–π interactions. These computational insights into the structure and chemistry of LbMTPSLs provide a foundation for future mechanistic studies and rational design to steer FPP cyclization pathways and significantly alter product profiles.
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