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The Pictet-Spengler reaction is a valuable route to 1,2,3,4-tetrahydro-β-carboline (THBC) and isoquinoline scaffolds found in many important pharmaceuticals. Strictosidine synthase (STR) catalyzes the Pictet-Spengler condensation of tryptamine and the aldehyde secologanin to give ( S )-strictosidine as a key intermediate in indole alkaloid biosynthesis. STRs also accept short-chain aliphatic aldehydes to give enantioenriched alkaloid products with up to 99% ee STRs are thus valuable asymmetric organocatalysts for applications in organic synthesis. The STR catalysis of reactions of small aldehydes gives an unexpected switch in stereopreference, leading to formation of the ( R )-products. Here we report a rationale for the formation of the ( R )-configured products by the STR enzyme from Ophiorrhiza pumila ( Op STR) using a combination of X-ray crystallography, mutational, and molecular dynamics (MD) studies. We discovered that short-chain aldehydes bind in an inverted fashion compared to secologanin leading to the inverted stereopreference for the observed ( R )-product in those cases. The study demonstrates that the same catalyst can have two different productive binding modes for one substrate but give different absolute configuration of the products by binding the aldehyde substrate differently. These results will guide future engineering of STRs and related enzymes for biocatalytic applications.

Radical S-adenosyl-l-methionine (SAM) enzymes are widely distributed and catalyze diverse reactions. SAM binds to the unique iron atom of a site-differentiated [4Fe-4S] cluster and is reductively cleaved to generate a 5'-deoxyadenosyl radical, which initiates turnover. 7-Carboxy-7-deazaguanine (CDG) synthase (QueE) catalyzes a key step in the biosynthesis of 7-deazapurine containing natural products. 6-Carboxypterin (6-CP), an oxidized analogue of the natural substrate 6-carboxy-5,6,7,8-tetrahydropterin (CPH 4 ), is shown to be an alternate substrate for CDG synthase. Under reducing conditions that would promote the reductive cleavage of SAM, 6-CP is turned over to 6-deoxyadenosylpterin (6-dAP), presumably by radical addition of the 5'-deoxyadenosine followed by oxidative decarboxylation to the product. By contrast, in the absence of the strong reductant, dithionite, the carboxylate of 6-CP is esterified to generate 6-carboxypterin-5'-deoxyadenosyl ester (6-CP-dAdo ester). Structural studies with 6-CP and SAM also reveal electron density consistent with the ester product being formed in crystallo. The differential reactivity of 6-CP under reducing and nonreducing conditions highlights the ability of radical SAM enzymes to carry out both polar and radical transformations in the same active site., Competing Interests: The authors declare no competing financial interest.

Lasso peptides are a class of bioactive ribosomally synthesized and post-translationally modified peptides (RiPPs), with a threaded knot structure that is formed by an isopeptide bond attaching the N-terminus of the peptide to a side chain carboxylate. Some lasso peptide biosynthetic clusters harbor an enzyme that specifically hydrolyzes the isopeptide bond to yield the linear peptide. We describe here the 2.4 Å resolution structure of a lasso peptide isopeptidase revealing a topologically novel didomain architecture consisting of an open β-propeller appended to an α/β hydrolase domain. The 2.2 Å resolution cocrystal structure of an inactive variant in complex with a lasso peptide reveals deformation of the substrate, and reorganization of the enzyme active site, which exposes and orients the isopeptide bond for hydrolysis. Structure-based mutational analysis reveals how this enzyme recognizes the lasso peptide substrate by shape complementarity rather than through sequence specificity. The isopeptidase gene can be used to facilitate genome mining, as a network-based mining strategy queried with this sequence identified 87 putative lasso peptide biosynthetic clusters, 65 of which have not been previously described. Lastly, we validate this mining approach by heterologous expression of two clusters encoded within the genome of Asticcaucalis benevestitus, and demonstrate that both clusters produce lasso peptides.

Lasso peptides are a class of ribosomally derived natural products with diverse bioactivities. The characteristic threaded lasso structure in these peptides derives from an isopeptide bond attaching the N-terminus of the peptide to an acidic side chain. Here we describe the heterologous expression of a lasso peptide gene cluster encoding two lasso peptides, astexin-2 and astexin-3, and solve the solution structure of astexin-3. This cluster also encodes an enzyme annotated as a protease. We show that this enzyme, AtxE2, is a lasso peptide isopeptidase that specifically hydrolyzes astexins-2 and -3, converting them to linear peptides. Astexin-3 is highly thermostable and resists unthreading after extensive heat treatment. In contrast, astexin-2 unthreads upon heat treatment. AtxE2 has no activity toward unthreaded astexin-2, demonstrating that this isopeptidase must recognize a knotted structure in order to function. We also use this isopeptidase as a tool to study evolutionary relationships between lasso peptide gene clusters.

Chemical methods for modifying proteins can enable studies aimed at uncovering biochemical function. Herein, we describe the use of thiol-ene coupling (TEC) chemistry to report on the function of branched (also referred to as forked) ubiquitin trimers. We show how site-specific isopeptide (Nε-Gly-L-homothiaLys) bonds are forged between two molecules of Ub, demonstrating the power of TEC in protein conjugation. Moreover, we demonstrate that the Nε-Gly-L-homothiaLys isopeptide bond is processed to a similar extent by deubiquitinases (DUBs) as that of a native Nε-Gly-L-Lys isopeptide bond, thereby establishing the utility of TEC in the generation of Ub-Ub linkages. TEC is then applied to the synthesis of branched Ub trimers. Interrogation of these branched derivatives with DUBs reveals that the relative orientation of the two Ub units has a dramatic impact on how they are hydrolyzed. In particular, cleavage of K48C-linkages is suppressed when the central Ub unit is also conjugated through K6C, whereas cleavage proceeds normally when the central unit is conjugated through either K11C or K63C. The results of this work presage a role for branched polymeric Ub chains in regulating linkage-selective interactions.

The Pictet-Spenglerase strictosidine synthase (STR1) has been recognized as a key enzyme in the biosynthesis of some 2000 indole alkaloids in plants, some with high therapeutic value. In this study, a novel function of STR1 has been detected which allows for the first time a simple enzymatic synthesis of the strictosidine analogue 3 harboring the piperazino[1,2-a]indole (PI) scaffold and to switch from the common tryptoline (hydrogenated carboline) to the rare PI skeleton. Insight into the reaction is provided by X-ray crystal analysis and modeling of STR1 ligand complexes. STR1 presently provides exclusively access to 3 and can act as a source to generate by chemoenzymatic approaches libraries of this novel class of alkaloids which may have new biological activities. Synthetic or natural monoterpenoid alkaloids with the PI core have not been reported before., (© 2011 American Chemical Society)

In this paper, we describe an optimized reconstitution of the thiamin thiazole synthase (ThiG) catalyzed reaction and demonstrate that the enzymatic product is an unanticipated dearomatized thiazole tautomer.