Christianson, D. W. | bioRxiv Science

bioRxiv2024

Engineering Substrate Channeling in Assembly-Line Terpene Biosynthesis

Fusicoccadiene synthase from P. amygdala (PaFS) is a bifunctional assembly-line terpene synthase containing a prenyltransferase domain that generates geranylgeranyl diphosphate (GGPP) from dimethylallyl diphosphate (DMAPP) and three equivalents of isopentenyl diphosphate (IPP), and a cyclase domain that converts GGPP into fusicoccadiene, a precursor of the diterpene glycoside Fusicoccin A. The two catalytic domains are linked by a flexible 69-residue polypeptide segment. The prenyltransferase domain mediates oligomerization to form predominantly octamers, and cyclase domains are randomly splayed out around the prenyltransferase core. Previous studies suggest that substrate channeling is operative in catalysis, since most of the GGPP formed by the prenyltransferase remains on the protein for the cyclization reaction. Here, we demonstrate that the flexible linker is not required for substrate channeling, nor must the prenyltransferase and cyclase domains be covalently linked to sustain substrate channeling. Moreover, substrate competition experiments with other diterpene cyclases indicate that the PaFS prenyltransferase and cyclase domains are preferential partners regardless of whether they are covalently linked or not. The cryo-EM structure of engineered "linkerless" construct PaFSLL, in which the 69-residue linker is spliced out and replaced with the tripeptide PTQ, reveals that cyclase pairs associate with all four sides of the prenyltransferase octamer. Taken together, these results suggest that optimal substrate channeling is achieved when a cyclase domain associates with the side of the prenyltransferase octamer, regardless of whether the two domains are covalently linked and regardless of whether this interaction is transient or locked in place.

biochemistry↗

bioRxiv2023

Crystal Structure of Histone Deacetylase 6 Complexed with (R)-Lipoic Acid, an Essential Cofactor in Central Carbon Metabolism

The enzyme cofactor (R)-lipoic acid plays a critical role in central carbon metabolism due to its catalytic function in the generation of acetyl-CoA, which links glycolysis with the tricarboxylic acid cycle. This cofactor is also essential for the generation of succinyl CoA within the tricarboxylic acid cycle. However, the biological functions of (R)-lipoic acid extend beyond metabolism owing to its facile redox chemistry. Most recently, the reduced form of (R)-lipoic acid, (R)-dihydrolipoic acid, has been shown to inhibit histone deacetylases (HDACs) with selectivity for the inhibition of HDAC6. Here, we report the 2.4 [A]-resolution X-ray crystal structure of the HDAC6-(R)-dihydrolipoic acid complex, and we report a dissociation constant (KD) of 350 nM for this complex as determined by isothermal titration calorimetry. The crystal structure illuminates key affinity determinants in the enzyme active site, including thiolate-Zn2+ coordination and S-{pi} interactions in the F583-F643 aromatic crevice. This study provides the first visualization of the connection between HDAC function and the biological response to oxidative stress: the dithiol moiety of (R)-dihydrolipoic acid can serve as a redox-regulated pharmacophore capable of simultaneously targeting the catalytic Zn2+ ion and the aromatic crevice in the active site of HDAC6.

biochemistry↗