Structure of methyltransferase RedM that forms the dimethylpyrrolinium of the bisindole reductasporine.

Bisindoles are biologically active natural products that arise from the oxidative dimerization of two molecules of l-tryptophan. In bacterial bisindole pathways, a core set of transformations is followed by the action of diverse tailoring enzymes that catalyze reactions that lead to diverse bisindole products. Among bisindoles, reductasporine is distinct due to its dimethylpyrrolinium structure. Its previously reported biosynthetic gene cluster encodes two unique tailoring enzymes, the imine reductase RedE and the dimethyltransferase RedM, which were shown to produce reductasporine from a common bisindole intermediate in recombinant E. coli. To gain more insight into the unique tailoring enzymes in reductasporine assembly, we reconstituted the biosynthetic pathway to reductasporine in vitro and then solved the 1.7 Å resolution structure of RedM. Our work reveals RedM adopts a variety of conformational changes with distinct open and closed conformations, and site-directed mutagenesis alongside sequence analysis identifies important active site residues. Finally, our work sets the stage for understanding how RedM evolved to react with a pyrrolinium scaffold and may enable the development of new dimethyltransferase catalysts.

An imine reductase that captures reactive intermediates in the biosynthesis of the indolocarbazole reductasporine.

Imine reductases (IREDs) and reductive aminases have been used in the synthesis of chiral amine products for drug manufacturing; however, little is known about their biological contexts. Here we employ structural studies and site-directed mutagenesis to interrogate the mechanism of the IRED RedE from the biosynthetic pathway to the indolocarbazole natural product reductasporine. Cocrystal structures with the substrate-mimic arcyriaflavin A reveal an extended active site cleft capable of binding two indolocarbazole molecules. Site-directed mutagenesis of a conserved aspartate in the primary binding site reveals a new role for this residue in anchoring the substrate above the NADPH cofactor. Variants targeting the secondary binding site greatly reduce catalytic efficiency, while accumulating oxidized side-products. As indolocarbazole biosynthetic intermediates are susceptible to spontaneous oxidation, we propose the secondary site acts to protect against autooxidation, and the primary site drives catalysis through precise substrate orientation and desolvation effects. The structure of RedE with its extended active site can be the starting point as a new scaffold for engineering IREDs and reductive aminases to intercept large substrates relevant to industrial applications.

Structure of the Oxygen, Pyridoxal Phosphate-Dependent Capuramycin Biosynthetic Protein Cap15.

Pyridoxal phosphate-dependent enzymes able to use oxygen as a co-substrate have emerged in multiple protein families. Here, we use crystallography to solve the 2.40 Å resolution crystal structure of Cap15, a nucleoside biosynthetic enzyme that catalyzes the oxidative decarboxylation of glycyl uridine. Our structural study captures the internal aldimine, pinpointing the active site lysine as K230 and showing the site of phosphate binding. Our docking studies reveal how Cap15 is able to catalyze a stereoselective deprotonation reaction, and bioinformatic analysis reveals active site residues that distinguish Cap15 from the structurally related d-glucosaminate-6-phosphate ammonia lyase and l-seryl-tRNA(Sec) selenium transferase (SelA). Our work provides the structural basis for further mechanistic investigation of a unique biosynthetic enzyme and provides a blueprint for understanding how oxygen reactivity emerged in the SelA-like protein family.

Biosynthesis of the N-N-Bond-Containing Compound l-Alanosine.

The formation of a N-N bond is a unique biochemical transformation, and nature employs diverse biosynthetic strategies to activate nitrogen for bond formation. Among molecules that contain a N-N bond, biosynthetic routes to diazeniumdiolates remain enigmatic. We here report the biosynthetic pathway for the diazeniumdiolate-containing amino acid l-alanosine. Our work reveals that the two nitrogen atoms in the diazeniumdiolate of l-alanosine arise from glutamic acid and aspartic acid, and we clarify the early steps of the biosynthetic pathway by using both in vitro and in vivo approaches. Our work demonstrates a peptidyl-carrier-protein-based mechanism for activation of the precursor l-diaminopropionate, and we also show that nitric oxide can participate in non-enzymatic diazeniumdiolate formation. Furthermore, we demonstrate that the gene alnA, which encodes a fusion protein with an N-terminal cupin domain and a C-terminal AraC-like DNA-binding domain, is required for alanosine biosynthesis.

An Asymmetric Reductase That Intercepts Acyclic Imino Acids Produced in Situ by a Partner Oxidase.

Acyclic imines are unstable in aqueous conditions. For this reason, known imine reductases, which enable the synthesis of chiral amines, mainly intercept stable cyclic imines. Here we report the detailed biochemical and structural characterization of Bsp5, an imino acid reductase from the d-2-hydroxyacid dehydrogenase family that reduces acyclic imino acids produced in situ by a partner oxidase. We determine a 1.6 Å resolution structure of Bsp5 in complex with d-arginine and coenzyme NADPH. Combined with mutagenesis work, our study reveals the minimal structural constraints for its biosynthetic activity. Furthermore, we demonstrate that Bsp5 can intercept more complex products from an alternate oxidase partner, suggesting that this oxidase-imino acid reductase pair could be evolved for biocatalytic conversion of l-amino acids to d-amino acids.

New reactions by pyridoxal phosphate-dependent enzymes.

Pyridoxal phosphate (PLP) is a cofactor that is widely employed in enzymology. This pyridine-containing cofactor can be used for reactions ranging from transaminations to oxidations. The catalytic versatility can be understood by considering the chemical features of this cofactor. In recent years, exciting new reactions involving PLP have been discovered in natural products biosynthesis, upending our understanding of what this cofactor is capable of. Here we review some of the most exciting PLP-dependent reactions from the last five years.