Genome mining for unknown–unknown natural products

Yee, Danielle A.; Niwa, Kanji; Perlatti, Bruno; Chen, Mengbin; Li, Yuqing; Tang, Yi

doi:10.1038/s41589-022-01246-6

Cited by 54 publications

(39 citation statements)

References 77 publications

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“…Most γ-synthases from the VGK subfamily catalyze γ-substitution using O-acetyl-L-homoserine (OAHS, 3) as the pro-electrophile, including secondary metabolic homologs. [14][15][16][17]33 Notably, E. coli and other γproteobacteria use O-succinyl-L-homoserine to reach the VGK intermediate, but also have low levels of promiscuous activity with 3. 13,33 Because the goal of this study was to experimentally distinguish between VGK subfamily functions, we assayed the 40 homologs for γ-synthase activity with Cys (1a) in competition with other thiol nucleophiles that have diverse physical properties (Figure 4).…”

Section: Resultsmentioning

confidence: 99%

“…More recently, members of the VGK subfamily have been shown to catalyze a range of powerful transformations in secondary metabolism, prompting further inquiry into their structure, mechanism, and function. [14][15][16][17] Sequence similarity network (SSN) analysis has emerged as a key tool for organizing the wealth of sequencing information by grouping enzymes into clusters based on sequence identity. 5 Analysis of VGK enzymes poses many challenges that are representative of the broader endeavor of enzyme functional annotation.…”

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confidence: 99%

“…More recently, pioneering work from the Tang Lab identified VGK enzymes that were originally annotated with a primary metabolic function instead play a role in secondary metabolism (Figure 1). [15][16][17] Notably, the enzymes CndF and Fub7 natively catalyze C-C bond formation and is an exciting reactivity to access new non-canonical amino acids (ncAAs). 15,16 Sequence ambiguity between primary and secondary metabolic VGK enzymes is further compounded by functional promiscuity.…”

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confidence: 99%

“…15,24 It is not known whether γ-lyases play a role in secondary metabolism and the known secondary metabolic γ-synthases are not reported to have promiscuous γ-lyase activity. [14][15][16][17] In this context, observation of a single catalytic activity is not sufficient to assign function within the VGK subfamily. Hence, new approaches will be needed to efficiently annotate the VGK subfamily with a high degree of function fidelity.…”

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confidence: 99%

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Multiplexed deconvolution of enzyme function in a PLP-dependent protein family

Zmich

Perkins

Bingman

et al. 2023

Preprint

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Sequence-based functional annotation of enzymes is an essential step in the discovery and development of new biocatalysts. The vinylglycine ketimine (VGK) subfamily of pyridoxal-phosphate dependent enzymes plays critical roles in sulfur metabolism and is home to a growing range of secondary metabolic enzymes that synthesize noncanonical amino acids. However, discovery of useful new enzymes has been slowed because functional assignments within the VGK sub-family are convoluted by pervasive mis-annotation. Here, we used a whole-cell substrate multiplexed screening approach to rapidly measure catalytic activities of 40 homologs in the VGK subfamily. This strategy gives direct information on enzyme specificity without having to purify each enzyme or measure individual kinetic constants. We identified a thermostable cystathionine γ-lyase from Thermobifida fusca and performed mechanistic and structural studies. For biocatalytic applications, we identified a well-behaved, thermostable, and promiscuous amino acid γ-synthase from Caldicellulosiruptor hydrothermalis (CahyGS). We showed CahyGS can catalyze a stereoselective γ-addition into L-allylglycine, providing preparative-scale access to a unique set of γ-branched amino acids. High resolution crystal structures of CahyGS show an open-closed transition associated with ligand binding and provide a basis for subsequent engineering. Together, these data show how multiplexed screening approaches aid in the rapid deconvolution of enzyme function and identify enzymes with useful properties for enzymology and biocatalysis.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Multiplexed deconvolution of enzyme function in a PLP-dependent protein family

Zmich

Perkins

Bingman

et al. 2023

Preprint

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“…Intermolecular nucleophilic addition at the γ-position of a vinyl glycine ketimine intermediate is precedented in PLP-dependent enzymes like cystathionine-γ-synthase (CGS) from methionine primary metabolism. 27 This ketimine intermediate has also been observed in specialized metabolite biosynthetic pathways, including: recently characterized fungal enzymes CndF, 28 Fub7, 29 and AnkD, 30 actinobacterial Mur24 involved in antibiotic nucleoside muraymycin biogenesis, 31 and indirectly through hydration in canavanine catabolism in select Gram-negative proteobacteria. 32 Although it remains to be biochemically characterized, LolC from the loline biosynthetic pathway is believed to proceed through an analogous ketimine intermediate with an L-proline nucleophile.…”

Section: Introductionmentioning

confidence: 99%

Mechanistic and structural insights into a divergent PLP-dependent L-enduracididine cyclase from a toxic cyanobacterium

Cordoza¹,

Chen

Blaustein³

et al. 2023

Preprint

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Cyclic arginine noncanonical amino acids (ncAAs) are found in several actinobacterial peptide natural products with therapeutically useful antibacterial properties. The preparation of ncAAs like enduracididine and capreomycidine currently takes multiple biosynthetic or chemosynthetic steps, thus limiting the commercial availability and applicability of these cyclic guanidine-containing amino acids. We recently discovered and characterized the biosynthetic pathway of guanitoxin, a potent freshwater cyanobacterial neurotoxin, that contains an arginine-derived cyclic guanidine phosphate within its highly polar structure. The ncAA L-enduracididine is an early intermediate in guanitoxin biosynthesis and is produced by GntC, a unique pyridoxal-5′-phosphate (PLP)-dependent enzyme. GntC catalyzes a cyclodehydration from a stereoselectively γ-hydroxylated L-arginine precursor via a reaction that functionally and mechanistically diverges from previously established actinobacterial cyclic arginine ncAA pathways. Herein, we interrogate L-enduracididine biosynthesis from the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024 using spectroscopic, stable isotope labeling techniques, and X-ray crystal structure-guided site-directed mutagenesis. GntC initially facilitates the reversible deprotonations of the α- and β- positions of its substrate prior to catalyzing an irreversible diastereoselective dehydration and subsequent intramolecular cyclization. The comparison of holo- and substrate bound GntC structures and activity assays on site-specific mutants further identified amino acid residues that contribute to the overall catalytic mechanism. These interdisciplinary efforts at structurally and functionally characterizing GntC enables an improved understanding of how Nature divergently produces cyclic arginine ncAAs and generates additional tools for their biocatalytic production and downstream biological applications.

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Evolutionary Spread of Distinct O‐methyltransferases Guides the Discovery of Unique Isoaspartate‐Containing Peptides, Pamtides

Lee,

Park,

Kim

et al. 2023

Advanced Science

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Ribosomally synthesized and post‐translationally modified peptides (RiPPs) are a structurally diverse class of natural products with a distinct biosynthetic logic, the enzymatic modification of genetically encoded precursor peptides. Although their structural and biosynthetic diversity remains largely underexplored, the identification of novel subclasses with unique structural motifs and biosynthetic pathways is challenging. Here, it is reported that peptide/protein L‐aspartyl O‐methyltransferases (PAMTs) present in several RiPP subclasses are highly homologous. Importantly, it is discovered that the apparent evolutionary transmission of the PAMT gene to unrelated RiPP subclasses can serve as a basis to identify a novel RiPP subclass. Biochemical and structural analyses suggest that homologous PAMTs convert aspartate to isoaspartate via aspartyl‐O‐methyl ester and aspartimide intermediates, and often require cyclic or hairpin‐like structures for modification. By conducting homology‐based bioinformatic analysis of PAMTs, over 2,800 biosynthetic gene clusters (BGCs) are identified for known RiPP subclasses in which PAMTs install a secondary modification, and over 1,500 BGCs where PAMTs function as a primary modification enzyme, thereby defining a new RiPP subclass, named pamtides. The results suggest that the genome mining of proteins with secondary biosynthetic roles can be an effective strategy for discovering novel biosynthetic pathways of RiPPs through the principle of “guilt by association”.

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Genome mining for unknown–unknown natural products

Cited by 54 publications

References 77 publications

Multiplexed deconvolution of enzyme function in a PLP-dependent protein family

Multiplexed deconvolution of enzyme function in a PLP-dependent protein family

Mechanistic and structural insights into a divergent PLP-dependent L-enduracididine cyclase from a toxic cyanobacterium

Evolutionary Spread of Distinct O‐methyltransferases Guides the Discovery of Unique Isoaspartate‐Containing Peptides, Pamtides

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