Deciphering the Biosynthetic Origin of l-allo-Isoleucine

Abstract

The nonproteinogenic amino acid L-allo-isoleucine (L-allo-Ile) is featured in an assortment of life forms comprised of, but not limited to, bacteria, fungi, plants and mammalian systems including Homo sapiens. Despite its ubiquity and functional importance, the specific origins of this unique amino acid have eluded characterization. In this study, we describe the discovery and characterization of two enzyme pairs consisting of a pyridoxal 5'-phosphate (PLP)-linked aminotransferase and an unprecedented isomerase synergistically responsible for the biosynthesis of L-allo-Ile from L-isoleucine (L-Ile) in natural products. DsaD/DsaE from the desotamide biosynthetic pathway in Streptomyces scopuliridis SCSIO ZJ46, and MfnO/MfnH from the marformycin biosynthetic pathway in Streptomyces drozdowiczii SCSIO 10141 drive L-allo-Ile generation in each respective system. In vivo gene inactivations validated the importance of the DsaD/DsaE pair and MfnO/MfnH pair in L-allo-Ile unit biosynthesis. Inactivation of PLP-linked aminotransferases DsaD and MfnO led to significantly diminished desotamide and marformycin titers, respectively. Additionally, inactivation of the isomerase genes dsaE and mfnH completely abolished production of all L-allo-Ile-containing metabolites in both biosynthetic pathways. Notably, in vitro biochemical assays revealed that DsaD/DsaE and MfnO/MfnH each catalyze a bidirectional reaction between L-allo-Ile and L-Ile. Site-directed mutagenesis experiments revealed that the enzymatic reaction involves a PLP-linked ketimine intermediate and uses an arginine residue from the C-terminus of each isomerase to epimerize the amino acid β-position. Consequently, these data provide important new insight into the origins of L-allo-Ile in natural products with medicinal potential and illuminate new possibilities for biotool development.

Solid-phase synthesis of cyclic hexapeptides wollamides A, B and desotamide B

“…This unique residue is found in other bioactive molecules such as mar-formycins, 7 and its biosynthetic origins have until recently been uncharacterized. 8 …”

Effects of Electrolytes on the Photocurrent of N‐Annulated Perylene‐Sensitized Photoelectrochemical Cells Based on NiO as Photocathode

“…Phosphate buffered solution (PBS) was generally used in photoelectrochemical measurements, [14,15,18] whereas the pH Figure 1. a) Absorption spectra of NPP and Co1 dissolved in THF and acetonitrile respectively, and the absorption spectra of NPP dye adsorbed on NiO film.…”

“…Of several of them the catalyst was dissolved in electrolyte [14] or coadsorbed in NiO film. [15,16] To facilitate electron transfer from dye molecule to catalyst, chromophores and noble metal free catalysts were linked by covalent, [17] non-covalent bonds, [18] layer by layer assembly. [19] Some of the reports introduced a Al 2 O 3 barrier layer by atomic layer deposition (ALD), effectively preventing the recombination or resisting the desorption of dye molecules.…”

“…[20] Sometimes p-DSPEC was connected in series with n-DSPEC. [15] But rarely reports have discussed what electrolyte means to the current density. Most of the p-DSPECs reported now employed PBS and acid Na 2 SO 4 aqueous solution as electrolyte.…”

Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity

“…The discovery [6][7][8] and engineering [9][10][11] of enzymes fueled by the formidable development in high-throughput biotechnology has allowed for tailored manufacturing of materials [12], medicines [13], and fine chemicals [14]. However, developing the full potential of biocatalysis and synthetic biology is dependent on exploring hitherto novel biochemistries [4,15] and reaction mechanisms [16] that expand the catalytic capabilities currently found in nature [15].…”