Discovery and Biocatalytic Application of a PLP-Dependent Amino Acid γ-Substitution Enzyme That Catalyzes C–C Bond Formation

Abstract

Pyridoxal phosphate (PLP)-dependent enzymes can catalyze various transformations of L-amino acids at ,  and  positions. These versatile enzymes are prominently involved in the biosynthesis of nonproteinogenic amino acids as building blocks of natural products, and are attractive biocatalysts. Here, we report the discovery of a two-step enzymatic synthesis of (2S, 6S)-6-methyl pipecolate 1, from the biosynthetic pathway of indole alkaloid citrinadin. The key enzyme CndF is PLP-dependent and catalyzes synthesis of (S)-2-amino-6-oxoheptanoate 3 that is in equilibrium with the cyclic Schiff base. The second enzyme CndE is a stereoselective imine reductase that gives 1. Biochemical characterization of CndF showed this enzyme performs -elimination of O-acetyl L-homoserine to generate the vinylglycine ketimine, which is subjected to nucleophilic attack by acetoacetate to form the new C-C bond in 3 and complete the -substitution reaction. CndF displays substrate promiscuity towards different -keto carboxylate and esters. Using a recombinant Aspergillus strain expressing CndF and CndE, feeding various alkyl--keto esters led to the biosynthesis of 6-substituted L-pipecolates. The discovery of CndF expands the repertoire of reactions that can be catalyzed by PLP-dependent enzymes.Nature is remarkable in building and using structurally diverse amino acids. 1-2 Nonproteinogeneic amino acids (NAAs), which constitute 96% of the naturally occurring amino acids, 1 are frequently incorporated into small molecules to broaden reactivity and to establish biologically relevant conformers. 3 Peptides that contain NAAs are less susceptible to proteolysis, thereby increasing the halflives during circulation. [4][5] The usefulness of NAAs have also been extensively explored through incorporation into recombinant proteins in Escherichia coli, yeast, and mammalian cells. 6 Because of these

Biosynthesis of Mycotoxin Fusaric Acid and Application of a PLP-Dependent Enzyme for Chemoenzymatic Synthesis of Substituted l-Pipecolic Acids

“…Retrobiosynthetic dissection of the γ–δ C–C bond in (2 S )-2-amino-5-formylnonanoic acid revealed this compound could be synthesized from OAH and n -hexanal ( 3 ) through an apparent γ-substitution reaction. Such reactions can be catalyzed by PLP-dependent enzymes, of which Fub7 is predicted to be (Figure D). To test this hypothesis, we purified recombinant Fub7 from E. coli and characterized its function in vitro (Figure S3).…”

“…The synthetic utility of Fub7 complements the recently discovered PLP-dependent enzyme CndF, which was used to prepare a suite of 6-alkyl-L-pipecolic acids. 11 Together with CndF, these PLP-dependent enzymes can be useful biocatalytic tools for the synthesis of an assortment of nitrogen-containing heterocycles.…”

“…Using the key C–C bond-forming enzyme Fub7 as a biocatalyst, we developed a facile method to prepare a panel of 5-alkyl-, 5,5-dialkyl-, and 5,5,6-trialkyl- l -pipecolic acids. The synthetic utility of Fub7 complements the recently discovered PLP-dependent enzyme CndF, which was used to prepare a suite of 6-alkyl- l -pipecolic acids . Together with CndF, these PLP-dependent enzymes can be useful biocatalytic tools for the synthesis of an assortment of nitrogen-containing heterocycles.…”

Enantioselective Dynamic Exchange Reactions of Imines

“…Using dynamic molecular systems to control molecular functions is receiving a great deal of attention from chemists . For example, pyridoxal 5-phosphate (PLP) is an indispensable and ubiquitous coenzyme used by living systems to participate in diverse reactions such as transamination, racemization, elimination, and other reactions, resulting in PLP being widely used as a biomimetic building block for the construction of dynamic molecular systems . A variety of PLP-dependent dynamic reactions originate from the unique molecular structure of PLP, particularly the presence of an o -OH group adjacent to the aldehyde group on the pyridine moiety.…”

Structural and Data Science-Driven Analysis to Assess Substrate Specificity of Diketopiperazine Reverse Prenyltransferase NotF: Cascade Biocatalytic Synthesis of (–)-Eurotiumin A

“…[51][52][53] Similarly, C2 reverse prenylation of cyclo(L-Trp-L-Val) ( 13) is a likely precursor to cristatumin F (S5), 54 cyclo(L-Trp-L-Trp) (35) to okaramine B (3, Figure 1), 19 and cyclo(L-Trp-L-Pip) (23) has been utilized as a synthetic intermediate to the marcfortines and related indole alkaloids (S10, Figure S1). 2,55,56 The ability to selectively C2 reverse prenylate these substrates therefore represents a solution to a key transformation within these biosynthetic systems. One-pot biocatalytic cascade to (-)-eurotiumin A.…”