Discovery, Structure, and Engineering of a cis‐Geranylfarnesyl Diphosphate Synthase

Abstract cis ‐Prenyltransferases ( cis ‐PTs) catalyze the sequential head‐to‐tail condensation of isopentenyl diphosphate (IPP) to allylic diphosphates, producing mixed E – Z prenyl diphosphates of varying lengths; however, the specific enzymes synthesizing cis ‐C 25 prenyl diphosphates have not been identified. Herein, we present the discovery and characterization of a cis ‐geranylfarnesyl diphosphate synthase (ScGFPPS) from Streptomyces clavuligerus . This enzyme demonstrates high catalytic proficiency in generating six distinct cis ‐polyisoprenoids, including three C 25 and three C 20 variants. We determined the crystal structure of ScGFPPS. Additionally, we unveil the crystal structure of nerylneryl diphosphate synthase (NNPS), known for synthesizing an all‐ cis ‐C 20 polyisoprenoid. Comparative structural analysis of ScGFPPS and NNPS has identified key differences that influence product specificity. Through site‐directed mutagenesis, we have identified eight single mutations that significantly refine the selectivity of ScGFPPS for cis ‐polyisoprenoids. Our findings not only expand the functional spectrum of cis ‐PTs but also provide a structural comparison strategy in cis ‐PTs engineering.