HAMA: a multiplexed LC-MS/MS assay for specificity profiling of adenylate-forming enzymes

Stanišić, Aleksa; Hüsken, Annika; Kries, Hajo

doi:10.1039/c9sc04222a

Cited by 32 publications

(47 citation statements)

References 42 publications

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“…We have shown that testing a small number of reversions to residue identities before swapping can generate significant improvements. 40 In this fashion, substrate specificity has been increased in the MS mutant and the temperature tolerance has been extended by 10 °C in the STAP mutant (Figure 3a). Since subdomain swapping only directly affects a limited number of interface residues, screening of reversion mutations comes at a low cost and may be more generally applicable to chimeric NRPS domains.…”

Section: Discussionmentioning

confidence: 89%

“…In the DKP formation assay used for screening, the STAP mutant showed a 6-fold increase in activity with slightly lower Val-selectivity (37%) and the MS mutant showed 2-fold higher activity at increased Val-selectivity (91%) compared to sdV-GrsA (54%, Supplementary Figure 6). 40 Thermal stability. Since sdV-GrsA is an unstable, chimeric protein impaired by engineering, we suspected improved structural integrity as a driver of evolutionary improvements.…”

Section: Resultsmentioning

confidence: 99%

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An Engineered Nonribosomal Peptide Synthetase Shows Opposite Amino Acid Loading and Condensation Specificity

Stanišić

Hüsken

Stephan

et al. 2021

Preprint

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<div> <p>Engineering of nonribosomal peptide synthetases (NRPS) has faced numerous obstacles despite being an attractive path towards novel bioactive molecules. Specificity filters in the nonribosomal peptide assembly line determine engineering success, but the relative contribution of adenylation (A-) and condensation (C-)domains is under debate. In the engineered, bimodular NRPS sdV-GrsA/GrsB1, the first module is a subdomain-swapped chimera showing substrate promiscuity. On sdV-GrsA and evolved mutants, we have employed kinetic modelling to investigate product specificity under substrate competition. Our model contains one step, in which the A-domain acylates the thiolation (T-)domain, and one condensation step deacylating the T-domain. The simplified model agrees well with experimentally determined acylation preferences and shows that the condensation specificity is mismatched with the engineered acylation specificity. Our model predicts changing product specificity in the course of the reaction due to dynamic T-domain loading, and that A-domain overrules C-domain specificity when T-domain loading reaches a steady-state. Thus, we have established a tool for investigating poorly accessible C-domain specificity through nonlinear kinetic modeling and gained critical insights how the interplay of A- and C-domains determines the product specificity of NRPSs.</p> </div>

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Section: Discussionmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

An Engineered Nonribosomal Peptide Synthetase Shows Opposite Amino Acid Loading and Condensation Specificity

Stanišić

Hüsken

Stephan

et al. 2021

Preprint

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“…In M. alpina, malpibaldin A (L-Phe derivative) is the major compound (17). Such discrepancies between adenylation preference and relative product formation rate may be caused by side-chain specificity of downstream reaction steps or differences in intracellular amino acid availability (51). Promiscuous adenylation leading to the parallel production of multiple products from one NRPS assembly line has been well studied in cyanobacterial enzymes and may be an important springboard for evolutionary diversification (45,54).…”

Section: Adenylation Domains Of Mpca and Mpba Activate L-amino Acidsmentioning

confidence: 99%

“…The hydroxamate formation assay was conducted as previously described (51). In brief, the assay was carried out at room temperature in 100 µL volume containing 50 S13).…”

Section: Multiplexed Hydroxamate Based Adenylation Domain Assay (Hama)mentioning

confidence: 99%

Bacterial-Like Nonribosomal Peptide Synthetases Produce Cyclopeptides in the Zygomycetous Fungus Mortierella alpina

Wurlitzer

Stanišić

Wasmuth

et al. 2021

Appl Environ Microbiol

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Fungi are traditionally considered as reservoir of biologically active natural products. However, an active secondary metabolism has long not been attributed to early diverging fungi such as Mortierella spec. Here, we report on the biosynthesis of two series of cyclic pentapeptides, the malpicyclins and malpibaldins, as products of Mortierella alpina ATCC32222. The molecular structures of malpicyclins were elucidated by HR-MS/MS, Marfey's method, and 1D and 2D NMR spectroscopy. In addition, malpibaldin biosynthesis was confirmed by HR-MS. Genome mining and comparative qRT-PCR expression analysis pointed at two pentamodular nonribosomal peptide synthetases (NRPS), malpicyclin synthetase MpcA and malpibaldin synthetase MpbA, as candidate biosynthetic enzymes. Heterologous production of the respective adenylation domains and substrate specificity assays proved promiscuous substrate selection and confirmed their respective biosynthetic roles. In stark contrast to known fungal NRPSs, MpbA and MpcA contain bacterial-like dual epimerase/condensation domains allowing the racemization of enzyme-tethered l-amino acids and the subsequent incorporation of d-amino acids into the metabolites. Phylogenetic analyses of both NRPS genes indicate a bacterial origin and a horizontal gene transfer into the fungal genome. We report on the as yet unexplored nonribosomal peptide biosynthesis in basal fungi which highlights this paraphylum as novel and underrated resource of natural products. IMPORTANCE Fungal natural compounds are industrially produced with application in antibiotic treatment, cancer medications and crop plant protection. Traditionally, higher fungi have been intensively investigated concerning their metabolic potential, but re-identification of already known compounds is frequently observed. Hence, alternative strategies to acquire novel bioactive molecules are required. We present the genus Mortierella as representative of the early diverging fungi as an underestimated resource of natural products. Mortierella alpina produces two families of cyclopeptides, denoted malpicyclins and malpibaldins, respectively, via two pentamodular nonribosomal peptide synthetases (NRPSs). These enzymes are much closer related to bacterial than to other fungal NRPSs, suggesting a bacterial origin of these NRPS genes in Mortierella. Both enzymes were biochemically characterized and are involved in as yet unknown biosynthetic pathways of natural products in basal fungi. Hence, this report establishes early diverging fungi as prolific natural compound producers and sheds light on the origin of their biosynthetic capacity.

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“…6 As a consequence, several strategies have been tested to edit the specificity of the A-domain. [7][8][9][10][11] Alternatively, domains and modules have been substituted and reshuffled. [12][13][14][15] One key emerging issue is the substrate tolerance of follow-up domains after changing the peptide sequence.…”

Section: Introductionmentioning

confidence: 99%

Engineered Nonribosomal Peptide Synthetase Shows Opposite Amino Acid Loading and Condensation Specificity

et al. 2021

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Engineering of nonribosomal peptide synthetases (NRPS) has faced numerous obstacles despite being an attractive path towards novel bioactive molecules. Specificity filters in the nonribosomal peptide assembly line determine engineering success, but the relative contribution of adenylation (A-) and condensation (C-)domains is under debate. In the engineered, bimodular NRPS sdV-GrsA/GrsB1, the first module is a subdomain-swapped chimera showing substrate promiscuity. On sdV-GrsA and evolved mutants, we have employed kinetic modelling to investigate product specificity under substrate competition. Our model contains one step, in which the A-domain acylates the thiolation (T-)domain, and one condensation step deacylating the T-domain. The simplified model agrees well with experimentally determined acylation preferences and shows that the condensation specificity is mismatched with the engineered acylation specificity. Our model predicts changing product specificity in the course of the reaction due to dynamic T-domain loading, and that A-domain overrules C-domain specificity when T-domain loading reaches a steady-state. Thus, we have established a tool for investigating poorly accessible C-domain specificity through nonlinear kinetic modeling and gained critical insights how the interplay of A-and C-domains determines the product specificity of NRPSs.File list (2) download file view on ChemRxiv Stanisic_manuscript_sdVGrsA .pdf (1.29 MiB) download file view on ChemRxiv Stanisic_Supplementary_Information_sdVGrsA.pdf (3.04 MiB)

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HAMA: a multiplexed LC-MS/MS assay for specificity profiling of adenylate-forming enzymes

Abstract: Adenylation enzymes are engineering targets in ribosomal and nonribosomal peptide synthesis. Through multiplexed LC-MS/MS measurement of hydroxamates, the HAMA assay records specificity profiles of these enzymes in a snap.

Cited by 32 publications

References 42 publications

An Engineered Nonribosomal Peptide Synthetase Shows Opposite Amino Acid Loading and Condensation Specificity

An Engineered Nonribosomal Peptide Synthetase Shows Opposite Amino Acid Loading and Condensation Specificity

Bacterial-Like Nonribosomal Peptide Synthetases Produce Cyclopeptides in the Zygomycetous Fungus Mortierella alpina

Engineered Nonribosomal Peptide Synthetase Shows Opposite Amino Acid Loading and Condensation Specificity

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