Functional convergence of structurally distinct thioesterases from cyanobacteria and plants involved in phylloquinone biosynthesis

Abstract

The synthesis of phylloquinone (vitamin K1) in photosynthetic organisms requires a thioesterase that hydrolyzes 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA) to release 1,4-dihydroxy-2-naphthoate (DHNA). Cyanobacteria and plants contain distantly related hotdog-fold thioesterases that catalyze this reaction, although the structural basis of these convergent enzymatic activities is unknown. To investigate this, the crystal structures of hotdog-fold DHNA-CoA thioesterases from the cyanobacterium Synechocystis (Slr0204) and the flowering plant Arabidopsis thaliana (AtDHNAT1) were determined. These enzymes form distinct homotetramers and use different active sites to catalyze hydrolysis of DHNA-CoA, similar to the 4-hydroxybenzoyl-CoA (4-HBA-CoA) thioesterases from Pseudomonas and Arthrobacter. Like the 4-HBA-CoA thioesterases, the DHNA-CoA thioesterases contain either an active-site aspartate (Slr0204) or glutamate (AtDHNAT1) that are predicted to be catalytically important. Computational modeling of the substrate-bound forms of both enzymes indicates the residues that are likely to be involved in substrate binding and catalysis. Both enzymes are selective for DHNA-CoA as a substrate, but this selectivity is achieved using divergent predicted binding strategies. The Slr0204 binding pocket is predominantly hydrophobic and closely conforms to DHNA, while that of AtDHNAT1 is more polar and solvent-exposed. Considered in light of the related 4-HBA-CoA thioesterases, these structures indicate that hotdog-fold thioesterases using either an active-site aspartate or glutamate diverged into distinct clades prior to the evolution of strong substrate specificity in these enzymes.

Isolation and characterization of mutants corresponding to the MENA, MENB, MENC and MENE enzymatic steps of 5′‐monohydroxyphylloquinone biosynthesis in Chlamydomonas reinhardtii

“…Altogether, this suggests that another thioesterase might operate in the PhQ biosynthesis pathway of green algae. Among the large family of thioesterases in C. reinhardtii , TEH4 (Cre07.g323150) is a possible candidate because it possesses the hot‐dog domain typical of DHNA–CoA thioesterase (Furt et al ., ), a putative binding site for coenzyme A, and a peroxisomal targeting sequence (PTS) (see below for further discussion). In flowering plants, genetic approaches identified the PHYLLO locus, which codes for a multi‐enzyme composed of four fused eubacterial men ‐homologous modules corresponding to MenF/MenD/MenC/MenH proteins, respectively (Gross et al ., ).…”

Unexpected roles of plastoglobules (plastid lipid droplets) in vitamin K 1 and E metabolism

“…How NDC1 is implicated in the AtmenG-de-pendent methylation step of phylloquinone synthesis remains a mystery. It will be interesting to see whether NDC1 is uniquely required for phylloquinone in Arabi-dopsis or whether this is also the case in other species [16,17,19].…”

“…Then, production of the DHNA-CoA is compartmentalized in peroxisomes where MenE, B and H act sequentially [16,17]. The pathway then returns to the plastid where the CoA moiety of DHNA-CoA is removed, and conversion to DHNA is catalyzed by a thioesterase hotdog-fold enzyme [19]. DHNA phytyltransferase (MenA) converts DHNA to Phytyl-NQ.…”

The renaissance of comparative biochemistry

“…C). Remarkably, both the cyanobacterial and plant/Lactobacillales thioesterases are selective for DHNA‐CoA as a substrate, though their selectivity is achieved using divergent binding strategies (Furt et al., ). Moreover, while the cyanobacterial‐type DHNA‐CoA thioesterase strictly uses DHNA‐CoA as the substrate (Widhalm et al., ), the Lactobacillales‐type DHNA‐CoA thioesterases in plants, and orthologs in Escherichia coli , are able to use a range of aromatic acyl‐CoA substrates (Widhalm et al., ; Chen et al., ).…”

“…Slr0204, cyanobacterial‐type DHNAT ; 4‐ HBT ‐ II , 4‐hydroxybenzoyl‐CoA thioesterase type II , plant/Lactobacillales‐type DHNAT . References indicated with subscripts are: a Gross et al., ; b Widhalm et al., ; c Widhalm et al., ; d Furt et al., ; e McCoy et al., .…”