Abstract:Significance Statement: Here we elucidate the full biosynthetic pathway of the fungal natural 40 product mycophenolic acid (MPA), which represents an unsolved mystery for decades. Besides the 41 intriguing enzymatic mechanisms, we reveal that the MPA biosynthetic enzymes are elegantly 42 compartmentalized; and the subcellular localization of the acyl-CoA hydrolase MpaH' in 43
“…The cavity specialized to accommodate MPA appears to be improper to accept other acyl-CoA esters to a right position for efficient hydrolysis. Supporting this, in our previous report, the k cat values of MpaH' for MPA-CoA were two orders of magnitude higher than the corresponding values for ten other CoA esters [6]. Moreover, as we predicted, MpaH' significantly hydrolyzed mycophenolate mofetil (MMF, i.e., 2-morpholinoethyl ester of MPA) [18], a clinically used MPA derivative, into MPA and 2-morpholinoethanol by acting as an esterase (Fig.…”
Section: Modeling Of Mpah' In Complex With Mpa-coasupporting
confidence: 74%
“…The preparation of the substrate MPA-CoA is the same as we reported before [6]. Briefly, 160 mg MPA was added to 3 mL acetonitrile and followed by the addition of 120 mg carbonyldiimidazole with stirring.…”
Section: Hydrolytic Assays Of Mpah'mentioning
confidence: 99%
“…the organelle-associated catalytic mechanisms [6]. In this pathway, the acyl-coenzyme A (CoA) hydrolase MpaH' that is subcellularly located in peroxisomes plays a key role in determining the specific production of MPA.…”
Mycophenolic acid (MPA) is a fungal natural product and first-line immunosuppressive drug for organ transplantations and autoimmune diseases. In the compartmentalized biosynthesis of MPA, the acyl-coenzyme A (CoA) hydrolase MpaH' located in peroxisomes catalyzes the highly specific hydrolysis of MPA-CoA to produce the final product MPA. The strict substrate specificity of MpaH' not only averts undesired hydrolysis of various cellular acyl-CoAs, but also prevents MPA-CoA from further peroxisomal b-oxidation catabolism. To elucidate the structural basis for this important property, in this study, we solve the crystal structures of the substrate-free form of MpaH' and the MpaH' S139A mutant in complex with the product MPA. The MpaH' structure reveals a canonical a/b-hydrolase fold with an unusually large cap domain and a rare location of the acidic residue D163 of catalytic triad after strand b6. MpaH' also forms an atypical dimer with the unique C-terminal helices a13 and a14 arming the cap domain of the other protomer and indirectly participating in the substrate binding. With these characteristics, we propose that MpaH' and its homologs form a new subfamily of a/b hydrolase fold protein. The crystal structure of MpaH' S139A /MPA complex and the modeled structure of MpaH'/ MPA-CoA, together with the structure-guided mutagenesis analysis and isothermal titration calorimetry (ITC) measurements, provide important mechanistic insights into the high substrate specificity of MpaH'.
“…The cavity specialized to accommodate MPA appears to be improper to accept other acyl-CoA esters to a right position for efficient hydrolysis. Supporting this, in our previous report, the k cat values of MpaH' for MPA-CoA were two orders of magnitude higher than the corresponding values for ten other CoA esters [6]. Moreover, as we predicted, MpaH' significantly hydrolyzed mycophenolate mofetil (MMF, i.e., 2-morpholinoethyl ester of MPA) [18], a clinically used MPA derivative, into MPA and 2-morpholinoethanol by acting as an esterase (Fig.…”
Section: Modeling Of Mpah' In Complex With Mpa-coasupporting
confidence: 74%
“…The preparation of the substrate MPA-CoA is the same as we reported before [6]. Briefly, 160 mg MPA was added to 3 mL acetonitrile and followed by the addition of 120 mg carbonyldiimidazole with stirring.…”
Section: Hydrolytic Assays Of Mpah'mentioning
confidence: 99%
“…the organelle-associated catalytic mechanisms [6]. In this pathway, the acyl-coenzyme A (CoA) hydrolase MpaH' that is subcellularly located in peroxisomes plays a key role in determining the specific production of MPA.…”
Mycophenolic acid (MPA) is a fungal natural product and first-line immunosuppressive drug for organ transplantations and autoimmune diseases. In the compartmentalized biosynthesis of MPA, the acyl-coenzyme A (CoA) hydrolase MpaH' located in peroxisomes catalyzes the highly specific hydrolysis of MPA-CoA to produce the final product MPA. The strict substrate specificity of MpaH' not only averts undesired hydrolysis of various cellular acyl-CoAs, but also prevents MPA-CoA from further peroxisomal b-oxidation catabolism. To elucidate the structural basis for this important property, in this study, we solve the crystal structures of the substrate-free form of MpaH' and the MpaH' S139A mutant in complex with the product MPA. The MpaH' structure reveals a canonical a/b-hydrolase fold with an unusually large cap domain and a rare location of the acidic residue D163 of catalytic triad after strand b6. MpaH' also forms an atypical dimer with the unique C-terminal helices a13 and a14 arming the cap domain of the other protomer and indirectly participating in the substrate binding. With these characteristics, we propose that MpaH' and its homologs form a new subfamily of a/b hydrolase fold protein. The crystal structure of MpaH' S139A /MPA complex and the modeled structure of MpaH'/ MPA-CoA, together with the structure-guided mutagenesis analysis and isothermal titration calorimetry (ITC) measurements, provide important mechanistic insights into the high substrate specificity of MpaH'.
“…3 iv). Overlapping in time frame with the work presented here, a preprint manuscript reported that the β -oxidation of primary metabolism was proposedly involved in the biosynthesis of 1 32 . However, the in vitro biochemical details required further investigation.Figure 3Reconstruction of 1 pathway in A. nidulans confirms two cleavage stages of the C15 prenyl side chain in 1 biosynthesis.…”
Mycophenolic acid (MPA, 1) and its derivatives are first-line immunosuppressants used in organ transplantation and for treating autoimmune diseases. Despite chemical synthetic achievements, the biosynthetic formation of a seven-carbon carboxylic acid pharmacophore side chain of 1, especially the processes involving the cleavage of the prenyl side chain between DHMP (4) and DMMPA (5), remains unknown. In this work, we identified a membrane-bound prenyltransferase, PgMpaA, that transfers FPP to 4 to yield FDHMP (6). Compound 6 undergoes the first cleavage step via a new globin-like enzyme PgMpaB to form a cryptic intermediate 12. Heterologous expression of PgMpa genes in Aspergillus nidulans demonstrates that the second cleavage step (from 12 to 5) of 1 is a PgMpa cluster-independent process in vivo. Our results, especially the discovery of the broad tolerance of substrates recognized by PgMpaB, set up a strategy for the formation of “pseudo-isopentenyl” natural products using fungal globin-like enzymes.
“…Some other sub-organelles, such as peroxisomes, lipid bodies, and vacuoles, are relatively orthogonal to the native essential process, making them ideal workhorses for metabolic compartmentalization. Furthermore, it has been showed that peroxisome compartmentalization of CoA-tailoring steps was required for efficient biosynthesis of penicillin (Meijer et al, 2010) and mycophenolic acid (Zhang et al, 2019b) in fungi, indicating that peroxisomes are ideal catalyzing houses for natural product biosynthesis. Peroxisomal targeting improved the production of fatty acid-derived molecules by up to 700% with relieved side pathway competition (Zhou et al, 2016;Zhu et al, 2017).…”
Natural products account for more than 50% of all small-molecule pharmaceutical agents currently in clinical use. However, low availability often becomes problematic when a bioactive natural product is promising to become a pharmaceutical or leading compound. Advances in synthetic biology and metabolic engineering provide a feasible solution for sustainable supply of these compounds. In this review, we have summarized current progress in engineering yeast cell factories for production of natural products, including terpenoids, alkaloids, and phenylpropanoids. We then discuss advanced strategies in metabolic engineering at three different dimensions, including point, line, and plane (corresponding to the individual enzymes and cofactors, metabolic pathways, and the global cellular network). In particular, we comprehensively discuss how to engineer cofactor biosynthesis for enhancing the biosynthesis efficiency, other than the enzyme activity. Finally, current challenges and perspective are also discussed for future engineering direction.
Production of Natural Products in YeastWith the recent advances of functional genomics, metabolic engineering, systems and synthetic biology, a plenty of natural products or their precursors have been heterologously synthesized in microbes especially My lab website: www.synbc. dicp.ac.cn Dedicated to the 70th anniversary of Dalian Institute of Chemical Physics, Chinese Academy of Sciences
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