Identification of a Hotdog Fold Thioesterase Involved in the Biosynthesis of Menaquinone in Escherichia coli

Abstract

Escherichia coli is used as a model organism for elucidation of menaquinone biosynthesis, for which a hydrolytic step from 1,4-dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) to 1,4-dihydroxy-2-naphthoate is still unaccounted for. Recently, a hotdog fold thioesterase has been shown to catalyze this conversion in phylloquinone biosynthesis, suggesting that its closest homolog, YbgC in Escherichia coli, may be the DHNA-CoA thioesterase in menaquinone biosynthesis. However, this possibility is excluded by the involvement of YbgC in the Tol-Pal system and its complete lack of hydrolytic activity toward DHNA-CoA. To identify the hydrolytic enzyme, we have performed an activity-based screen of all nine Escherichia coli hotdog fold thioesterases and found that YdiI possesses a high level of hydrolytic activity toward DHNA-CoA, with high substrate specificity, and that another thioesterase, EntH, from siderophore biosynthesis exhibits a moderate, much lower DHNA-CoA thioesterase activity. Deletion of the ydiI gene from the bacterial genome results in a significant decrease in menaquinone production, which is little affected in ⌬ybgC and ⌬entH mutants. These results support the notion that YdiI is the DHNA-CoA thioesterase involved in the biosynthesis of menaquinone in the model bacterium.

Genomic, Proteomic, and Biochemical Analysis of the Organohalide Respiratory Pathway in Desulfitobacterium dehalogenans

“…The genome encodes a complete menaquinone synthesis pathway (menFDHCEBIAG) (43). We found nine flavoproteins resembling FAD binding proteins present in a wide range of bacteria, often associated with electron transfer or reduction of terminal electron acceptors.…”

Functional Screening and In Vitro Analysis Reveal Thioesterases with Enhanced Substrate Specificity Profiles That Improve Short-Chain Fatty Acid Production in Escherichia coli

“…The disparity between the specific activities of Fs2108 for these two substrates indicates that our pathway relies on the promiscuous thioesterase activity of Fs2108. This is particularly evident for two thioesterases, EcYdiI and Pr1697, that were associated with increased valerate production despite having much higher specific activity for long-chain acyl-CoAs in the case of Pr1687 or aromatic acyl-CoAs for EcYdiI (34). The fact that all six thioesterases that were investigated in vitro prefer alternate substrates over those provided in our pathways suggests that both protein engineering and future bioprospecting efforts could further improve on the short-chain fatty acyl-CoA thioesterases discovered here.…”

“…EcYdiI had low activity for all substrates tested. A recent publication showing that EcYdiI has strong activity on the aromatic compound 1,4-dihydroxy-2-naphthoyl-CoA provides justification for the weak activity of this enzyme on the substrates provided in this study (34).…”

“…In our recombinant strains, EcYdiI and the other screened thioesterases compete with the downstream pathway enzyme Ter for the substrate trans-2-pentenoyl-CoA. One plausible explanation for the absence of detectable trans-2-pentenoate from this recombinant strain is that EcYdiI has a K m , a measure of substrate affinity, for both trans-2-pentenoyl-CoA and valeryl-CoA greater than the 20 M concentration used in our in vitro assay, which is reasonable to assume because EcYdiI has evolved for specificity toward aromatic acids (34). If Ter has a lower K m for trans-2-pentenoyl-CoA than EcYdiI, then it could reduce the intracellular concentration of trans-2-pentenoyl-CoA below 20 M, causing EcYdiI activity for this compound to become physiologically irrelevant.…”

Discovery of Bicyclic Inhibitors Against Menaquinone Biosynthesis

“…Both routes begin with chorismate; however, the precursors for MK biosynthesis are different in both pathways. In the classical MK pathway, 1,4-dihydroxy-2-naphthoate is derived from chorismate by Men FDHCEB enzymes and MenI [5] is converted to an unmethylated MK by 1,4-dihydroxy-2-naphthoate prenyltransferase (MenA), followed by methylation by MenG/UbiE to produce the final product MK. In the alternative futalosine pathway, MK is biosynthesized from 1,4-dihydroxy-6-napthoate, although unknown enzymes catalyze the final reaction [6].…”