Supplemental Nicotinamide Dose-Dependently Regulates Body Phosphorus Excretion via Altering Type II Sodium-Phosphate Co-Transporter Expressions in Laying Hens.

Background: Dietary supplemental nicotinamide is used to treat hyperphosphatemia in humans. However, the mechanisms of its impact on body phosphorus homeostasis remain unclear.

Objective: This study was to determine effects and molecular mechanisms of 3 dietary nicotinamide concentrations on body phosphorus homeostasis in laying hens.

Protein engineering of oxidosqualene-lanosterol cyclase into triterpene monocyclase.

A computational modeling/protein engineering approach was applied to probe H234, C457, T509, Y510, and W587 within Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase (ERG7), which spatially affects the C-10 cation of lanosterol formation. Substitution of Trp587 to aromatic residues supported the "aromatic hypothesis" that the π-electron-rich pocket is important for the stabilization of electron-deficient cationic intermediates. The Cys457 to Gly and Thr509 to Gly mutations disrupted the pre-existing H-bond to the protonating Asp456 and the intrinsic His234 : Tyr510 H-bond network, respectively, and generated achilleol A as the major product.

The cysteine 703 to isoleucine or histidine mutation of the oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae generates an iridal-type triterpenoid.

The Cys703 to Ile or His mutation within Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase ERG7 (ERG7(C703I/H)) generates an unusual truncated bicyclic rearranged intermediate, (8R,9R,10R)-polypoda-5,13E,17E,21-tetraen-3β-ol, related to iridal-skeleton triterpenoid. Numerous oxidosqualene-cyclized truncated intermediates, including tricyclic, unrearranged tetracyclic with 17α/β exocyclic hydrocarbon side chain, rearranged tetracyclic, and chair-chair-chair tricyclic intermediates (compounds 3-9), were also isolated from the ERG7(C703X) site-saturated mutations or the ERG7(F699T/C703I) double mutation, indicating the functional role of the Cys703 residue in stabilizing the bicyclic C-8 cation and the rearranged intermediate or interacting with Phe699, and opened a new avenue of engineering ERG7 for producing biological active agents.

Mutation of isoleucine 705 of the oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae affects lanosterol's C/D-ring cyclization and 17α/β-exocyclic side chain stereochemistry.

Site-saturated substitution in Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase at Ile705 position produced three chair-boat-chair (C-B-C) truncated tricyclic compounds, two 17α-exocyclic protosteryl intermediates, two protosteryl C-17 truncated rearranged intermediates and the normal biosynthetic product, lanosterol. These results indicated the importance of the Ile705 residue in affecting lanosterol's C/D ring stabilization including 6-6-5 tricyclic and protosteryl C-17 cations and 17α/β-exocyclic side chain stereochemistry.

Alteration of the substrate's prefolded conformation and cyclization stereochemistry of oxidosqualene-lanosterol cyclase of Saccharomyces cerevisiae by substitution at phenylalanine 699.

The Saccharomyces cerevisiae ERG7(Phe699) mutants produced one chair-chair-chair (C-C-C) and two chair-boat-chair (C-B-C) truncated tricyclic compounds, one tetracyclic 17alpha-exocyclic unrearranged intermediate, and two 17beta-exocyclic truncated rearranged intermediates. These results provided direct evidence for the importance of the residue in affecting mechanistic transitions between C-B-C and C-C-C substrate conformation and between the 17alpha- and 17beta-exocyclic side chain stereochemistry as well as in stabilizing the 6-6-5 tricyclic and the protosteryl C-17 cations.

Protein plasticity: a single amino acid substitution in the Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase generates protosta-13(17),24-dien-3beta-ol, a rearrangement product.

To provide insights into the structure-function relationships of oxidosqualene-lanosterol cyclase (ERG7) from Saccharomyces cerevisiae, the Phe699 was exchanged against hydrophilic polar uncharged residues Ser, Thr, Cys, Gln, and Tyr to characterize its product profile and functional role in ERG7 activity. Among the substitutions, only the ERG7(F699T) mutant produced novel protosta-13(17),24-dien-3beta-ol as the sole truncated rearrangement product. The results suggest that Phe699Thr mutation is likely to affect the C-17 cation stabilization during the rearrangement process.