Structural Basis and Enzymatic Mechanism of the Biosynthesis of C9‐ from C10‐Monoterpenoid Indole Alkaloids

Cutting carbons : The three‐dimensional structure of polyneuridine aldehyde esterase (PNAE) gives insight into the enzymatic mechanism of the biosynthesis of C₉‐ from C₁₀‐monoterpenoid indole alkaloids (see scheme). PNAE is a very substrate‐specific serine esterase. It harbors the catalytic triad S87‐D216‐H244, and is a new member of the α/β‐fold hydrolase superfamily. Its novel function leads to the diversification of alkaloid structures.

     

Converting an Esterase into an Epoxide Hydrolase

Entering the fold : A common structural motif in hydrolytic enzymes is the α,β‐hydrolase fold. The interconversion of one enzyme into another by introduction of mechanistically important residues is not enough; only substitution of a loop allows epoxide hydrolase activity in the esterase scaffold to be formed (see picture; structure comparison of epoxide hydrolases (green) with the esterase (orange)). The result is an enantioselective chimeric enzyme.

     

Sequence-Based Prediction of Promiscuous Acyltransferase Activity in Hydrolases

Certain hydrolases preferentially catalyze acyl transfer over hydrolysis in an aqueous environment. However, the molecular and structural reasons for this phenomenon are still unclear. Herein, we provide evidence that acyltransferase activity in esterases highly correlates with the hydrophobicity of the substrate-binding pocket. A hydrophobicity scoring system developed in this work allows accurate prediction of promiscuous acyltransferase activity solely from the amino acid sequence of the cap domain. This concept was experimentally verified by systematic investigation of several homologous esterases, leading to the discovery of five novel promiscuous acyltransferases. We also developed a simple yet versatile colorimetric assay for rapid characterization of novel acyltransferases. This study demonstrates that promiscuous acyltransferase activity is not as rare as previously thought and provides access to a vast number of novel acyltransferases with diverse substrate specificity and potential applications.     

Two efficient ways to 2-O- and 5-O-feruloylated 4-nitrophenyl α-l-arabinofuranosides as substrates for differentiation of feruloyl esterases

4-Nitrophenyl 2-O-(E)-feruloyl-α-l-arabinofuranoside 1 and 4-nitrophenyl 5-O-(E)-feruloyl-α-l-arabinofuranoside 2 have been synthesized by two different routes. Monoferuloylation was accomplished by a chemoenzymatic sequence employing a regioselective transesterification catalyzed by lipases. The feruloyl group was introduced to enzymatically prepared 2,3- and 3,5-diacetates of 4-nitrophenyl α-l-arabinofuranoside by reaction with 4-O-acetylferuloyl chloride. Removal of the protecting acetyl groups yielded 1 and 2. An alternative chemical synthesis suitable for preparation of larger quantities of 1 and 2 also is presented. The new substrates represent convenient tools to differentiate feruloyl esterases on the basis of their substrate specificity.     

Biotransformation of coal substructure model compounds by microbial enzymes

Research with both coal substructure model compounds and macromolecular coal has shown that intermonomeric chemical bonds within coal are susceptible to cleavage by microbial enzymes from bacterial and fungal sources. This is particularly true when low rank lignite coals are used as substrates for enzymes after first being solubilized under alkaline conditions to form water-soluble coal polymers. When these soluble polymers are used as substrates, high-performance liquid chromatography (HPLC) methods are used to show that particular enzymes catalyze their depolymerization. By using chemical presolubilization followed by enzymatic depolymerization, it may be possible to develop commercial processes for the enzymatic depolymerization of coal into useful low-mol-wt chemicals, or into liquid or gaseous fuels. Evidence indicates that oxidative enzymes, such as peroxidases or etherases, and hydrolytic enzymes, such as esterases, have the best potential for effectively depolymerizing coal. Recent findings in our laboratory also suggest that certain hydrolases from saprophytic soil fungi may also work well.     

Synthesis and Biological Evaluation of Water-Soluble Esterase-Activated CO-Releasing Molecules Targeting Mitochondria

Due to the beneficial effects of carbon monoxide as a cell-protective and anti-inflammatory agent, CO-releasing molecules (CORMs) offer some promising potential applications in medicine. In this context, we synthesized a set of acyloxy-cyclohexadiene-Fe(CO)₃ complexes, all displaying a N-methyl-pyridinium triflate moiety in the ester side chain, as mitochondria-targeting esterase-triggered CORM prodrugs. Whereas the compounds in which the acyloxy substituent is attached to the 2-position of the diene-Fe(CO)₃ unit (A series) spontaneously release CO upon dissolution in phosphate buffer, which remarkably is partly suppressed in the presence of porcine liver esterase (PLE), the 1-substituted isomers (B series) show the expected PLE-induced release of CO (up to 3 equiv.). The biological activity of Mito-CORMs 2 / 3 - B and their isophorone-derived analogs 2/3 - A ’, which also displayed PLE-induced CO release, was assessed by using human umbilical vein endothelial cells (HUVEC). Whereas Mito-CORMs 2/3 - B were not cytotoxic up to 500 μM (MTT assay), Mito-CORMs 2 / 3 - A ’ caused significant toxicity at concentrations above 50 μM. The anti-inflammatory potential of both Mito-CORM variants was demonstrated by concentration-dependent down-regulation of the pro-inflammatory markers VCAM-1, ICAM-1 and CXCL1 as well as induction of HO-1 in TNFα-stimulated human umbilical vein endothelial cells (HUVECs; western blotting and qPCR). Energy phenotyping by seahorse real-time cell metabolic analysis, revealed opposing shifts of metabolic potentials in cells treated either with Mito-CORMs 2/3 - B (increased mitochondrial respiration and glycolytic activity) or Mito-CORMs 2/3 - A ’ (suppressed mitochondrial respiration and increased glycolytic activity). Thus, the Mito-CORMs represent valuable tools for the safe and targeted delivery of CO to mitochondria as a subcellular compartment to induce positive anti-inflammatory effects with only minor shifts in cellular energy metabolism. Also, due to their water solubility, these compounds provide a promising starting point for further pharmacological studies.