Identification of a crucial amino acid implicated in the hydroxylation/desaturation ratio of CpFAH12 bifunctional hydroxylase

Abstract

Claviceps purpurea bifunctional Δ12‐hydroxylase/desaturase, CpFAH12, and monofunctional desaturase CpFAD2, share 86% of sequence identity. To identify the underlying determinants of the hydroxylation/desaturation specificity, chimeras of these two enzymes were tested for their fatty acid production in an engineered Yarrowia lipolytica strain. It reveals that transmembrane helices are not involved in the hydroxylation/desaturation specificity whereas all cytosolic domains have an impact on it. Especially, replacing the CpFAH12 cytosolic part near the second histidine‐box by the corresponding CpFAD2 part annihilates all hydroxylation activity. Further mutagenesis experiments within this domain identified isoleucine 198 as the crucial element for the hydroxylation activity of CpFAH12. Monofunctional variants performing the only desaturation were obtained when this position was exchanged by the threonine of CpFAD2. Saturation mutagenesis at this position showed modulation in the hydroxylation/desaturation specificity in the different variants. The WT enzyme was demonstrated as the most efficient for ricinoleic acid production and some variants showed a better desaturation activity. A model based on the recently discovered membrane desaturase structures indicate that these changes in specificity are more likely due to modifications in the di‐iron center geometry rather than changes in the substrate binding mode.

A Desaturase‐Like Enzyme Catalyzes Oxazole Formation in Pseudomonas Indolyloxazole Alkaloids

“…Zuschriften 8864 www.angewandte.de of two adjacent hydrogens in an O 2 -dependent process from carbon centers to generate an olefinic moiety, [26] but hydroxylation reactions are also known, [27] and even small structural changes can alter the reactivity and product outcome. [28] The assumed initial reaction is hydrogen abstraction to create a short-lived carbon-centered radical, but details of the mechanism have not been entirely clarified. [29][30][31] Theoretical investigations disfavor the formation of a carbocation that has been discussed as an alternative reactive intermediate.…”

“…IoxB features the conserved tripartite motif of eight histidines, essential for catalytic function in membrane desaturases (Supporting Information, Figure S4) [25] . A common reaction of desaturases is the removal of two adjacent hydrogens in an O 2 ‐dependent process from carbon centers to generate an olefinic moiety, [26] but hydroxylation reactions are also known, [27] and even small structural changes can alter the reactivity and product outcome [28] . The assumed initial reaction is hydrogen abstraction to create a short‐lived carbon‐centered radical, but details of the mechanism have not been entirely clarified [29–31] .…”

A Desaturase‐Like Enzyme Catalyzes Oxazole Formation in Pseudomonas Indolyloxazole Alkaloids

“…Communications of two adjacent hydrogens in an O 2 -dependent process from carbon centers to generate an olefinic moiety, [26] but hydroxylation reactions are also known, [27] and even small structural changes can alter the reactivity and product outcome. [28] The assumed initial reaction is hydrogen abstraction to create a short-lived carbon-centered radical, but details of the mechanism have not been entirely clarified. [29][30][31] Theoretical investigations disfavor the formation of a carbocation that has been discussed as an alternative reactive intermediate.…”

Metabolic Engineering for Unusual Lipid Production in Yarrowia lipolytica

“…When the strain also overexpressed FAH12 from C. purpurea, RA titers reached 12 g/L (representing 60% of total lipids) in a bioreactor. Recently, secondary structure prediction and mutagenesis helped identify the crucial domain involved in CpFAH12 hydroxylation in Y. lipolytica [99] and a consolidated RA production process was developed by introducing RA synthesis in a strain engineered for cellulose utilization [100].…”