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Chiral amine moieties are widely distributed in bioactive natural products and pharmaceutical ingredients. NAD(P)H-dependent imine reductases (IREDs) have been identified as potential biocatalysts for chiral amine synthesis via asymmetric reduction of the imine substrates. In this work, I characterized two unusual imino-acid-reducing enzymes, Punc5 and Bsp5, from the D-2-hydroxy-acid dehydrogenase (DHDH) family. The DHDH enzymes are known for reducing α-keto acids directly to the corresponding chiral hydroxy acids; however, both Punc5 and Bsp5 demonstrate imine reductase activity. Specifically, when coupled with L-arginine oxidase Ind4, both enzymes can use the coenzyme NAD(P)H to stereo-specifically reduce the Ind4 products didedydroarginine and dedydroarginine to D-4,5-dehydroarginine and D-arginine, respectively. Furthermore, Punc5 shows a DHDH activity, converting 2-ketoarginine to 5-guanidino-2-hydroxypentanoic acid. Both IREDs and DHDHs belong to the NAD(P)H-dependent oxidoreductase family; however, imine reduction catalyzed by DHDHs had never been reported before. To understand how Punc5 and Bsp5 evolved from DHDHs with asymmetric imino-acid-reducing activities, and to offer insights into NAD(P)H-dependent oxidoreductases’ chemoselectivity, I obtained ~1.6 Å resolution ternary structures of each enzyme bound with coenzyme NADPH and product D-arginine. These ternary structures of Punc5 and Bsp5 at high resolution closely resemble typical DHDHs; however, the spatial relationship of the coenzyme, product, and catalytic residues within the active site suggests a different catalytic mechanism from typical DHDHs. Structure-guided mutagenesis work uncovered an essential residue Tyr97 for substrate binding in Punc5. Biochemical characterization of the Punc5-Y97F variant suggests imine reduction under the acidic condition is a more facile reaction compared to ketone reduction as Punc5-Y97F is active towards imino acids, but it is inactive towards 2-ketoarginine.This unique imino-acid-reducing activity demonstrated by Punc5 and Bsp5 indicate that other subfamilies of NAD(P)H-dependent oxidoreductases besides known IREDs could also have the potential to produce chiral amines and be applied in pharmaceutical industry. Besides, our work offered three-dimensional frameworks for understanding how these unusual imino acid reductases differ from typical DHDHs, setting the stage for further engineering efforts to either enhance their catalytic efficiency or expand their substrate scopes.
Cladoniamides are a set of bisindole compounds that contain an indolotryptoline rather than the more common indolocarbazole scaffold. Besides their interesting structures, several of the cladoniamides have been found to be potent cytotoxic agents. We set out to isolate the brominated analogues of known cladoniamides by supplementing the fermentation medium with KBr, which led to the production of 5-bromocladoniamide A. However, the observed production levels were very low. To determine whether the selection against the bromo-substrates is early or late in the cladoniamide biosynthetic pathway, we synthesized 3-chloroarcyriaflavin and 3-bromoarcyriaflavin. These substrates were then fed into Streptomyces albus + cla (ΔclaC), which contains the complete cladoniamide biosynthetic pathway, except one crucial gene required for the production of cladoniamides. Through the feeding experiment, we found approximately equal amount of incorporation of the chloro and bromo substrates. The results suggest that the substrate selectivity against bromo precursors is upstream in the pathway from the enzyme encoded by the inactivated gene. Overall, we have observed the production of brominated cladoniamides through the two different methods of modifying the growth conditions and of precursor directed biosynthesis. Furthermore, this work presents a facile way to generate new indolotryptoline molecules through synthetic generation of desired indolocarbazole substrates and then biological conversion using the cladoniamide biosynthetic pathway.