Canavanine utilization via homoserine and hydroxyguanidine by a PLP-dependent γ-lyase in Pseudomonadaceae and Rhizobiales

Hauth, Franziskus; Buck, Hiltrun; Stanoppi, Marco; Hartig, Jörg S.

doi:10.1039/d2cb00128d

Cited by 12 publications

(9 citation statements)

References 69 publications

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“…Elimination of Lhomoserine (Hse, 4) and L-canavanine (Cnv, 5) has been previously reported for enzymes of this subfamily. 23,39,40 We also wanted to test the ability of enzymes to eliminate the dimethyl sulfonium analog of Met, S-Me-Met (6). 1 undergoes an O-N acyl shift at basic pH and deacetylates, whereas 6 is water stable and easy to synthesize.…”

Section: Resultsmentioning

confidence: 99%

“…20 Lastly, there are other VGK γ-lyases can catabolize Met, Hcy, and L-canavanine (Cnv). [21][22][23] Each of the above primary metabolic pathways features a distinct VGK enzyme, but their high degree of sequence identity has precluded differentiation based on sequence alone. 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).…”

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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%

mentioning

confidence: 99%

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

Zmich

Perkins

Bingman

et al. 2023

Preprint

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“…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. Overall, due to its unique sequence homology, divergent chemical reaction, and important role in guanitoxin biosynthesis, we sought to determine the mechanism of GntC-mediated cyclization, rationalize its strict diastereoselectivity and understand structural features responsible for its catalytic function.…”

Section: Introductionmentioning

confidence: 99%

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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“…Intermolecular nucleophilic addition at the γ-position of a vinylglycine ketimine intermediate is precedented in PLP-dependent enzymes like cystathionine-γ-synthase (CGS) from methionine primary metabolism . This ketimine intermediate has also been observed in specialized metabolite biosynthetic pathways, including recently characterized fungal enzymes CndF, Fub7, and AnkD, actinobacterial Mur24 involved in antibiotic nucleoside muraymycin biogenesis, and indirectly through hydration in canavanine catabolism in select Gram-negative proteobacteria . 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

et al. 2023

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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 spectroscopy, stable isotope labeling techniques, and X-ray crystallography structure-guided site-directed mutagenesis. GntC initially facilitates the reversible deprotonations of the α- and β-positions of its substrate before 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 enable an improved understanding of how nature divergently produces cyclic arginine ncAAs and generate additional tools for their biocatalytic production and downstream biological applications.

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Canavanine utilization via homoserine and hydroxyguanidine by a PLP-dependent γ-lyase in Pseudomonadaceae and Rhizobiales

Abstract: A novel degradation pathway enables rhizosphere-associated bacteria to utilize canavanine.

Cited by 12 publications

References 69 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

Mechanistic and Structural Insights into a Divergent PLP-Dependent l-Enduracididine Cyclase from a Toxic Cyanobacterium

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