Solvent Engineering Substantially Enhances the Chemoenzymatic Production of Surfactin

Wagner, Björn; Sieber, Stephan A.; Baumann, Marcus; Marahiel, Mohamed A.

doi:10.1002/cbic.200500495

Cited by 12 publications

(10 citation statements)

References 45 publications

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“…Support for this hypothesis was provided by studying the in vitro cyclization catalysed by the P26G mutant of SrfTE in organic solvents like N,Ndimethylformamide. 18 Under these conditions, where water was excluded from the reaction medium, the P26G mutant catalysed only the macrocyclization and not the hydrolysis of the linear surfactin peptide.…”

Section: The Pmsf-inhibited Form Of Fentementioning

confidence: 99%

“…Reactions were performed at room temperature and quenched by the addition of 4% (v/v) TFA. Products were analysed by HPLC-MS on a reversed-phase C 18 Nucleodur column 125/3 (Macherey and Nagel) with a flow-rate of 0.8 ml min K1 at 40 8C. The gradient applied was 20%-60% MeCN (with 0.1% TFA) in water (with 0.1% TFA) over 15 min.…”

Section: Fente Cyclization Assay and Determination Of Kinetic Parametersmentioning

confidence: 99%

See 1 more Smart Citation

The Thioesterase Domain of the Fengycin Biosynthesis Cluster: A Structural Base for the Macrocyclization of a Non-ribosomal Lipopeptide

Samel

Wagner

Marahiel

et al. 2006

Journal of Molecular Biology

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123

127

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Section: The Pmsf-inhibited Form Of Fentementioning

confidence: 99%

Section: Fente Cyclization Assay and Determination Of Kinetic Parametersmentioning

confidence: 99%

The Thioesterase Domain of the Fengycin Biosynthesis Cluster: A Structural Base for the Macrocyclization of a Non-ribosomal Lipopeptide

Samel

Wagner

Marahiel

et al. 2006

Journal of Molecular Biology

Self Cite

123

127

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“…These two solutions were mixed and shaken over night at room temperature and dried in Speed-Vac at 45°C. The C-terminal thioester activation was performed following Wagner's method (Wagner et al 2006). 12 lmol DCC, 60 lmol HOBT, 60 lmol N-acetylcysteamine in 500 ll THF were added into the Boc-protected LHB.…”

Section: Chemicals and Synthesis Of Lhb And Lhb-snacmentioning

confidence: 99%

An in vitro system to study cyclopeptide heterophyllin B biosynthesis in the medicinal plant Pseudostellaria heterophylla

Zhu

Tan

et al. 2011

Plant Cell Tiss Organ Cult

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Plant cyclopeptides are a large group of small molecule metabolites found in a wide variety of plants, including traditional Chinese medicinal plants. Many of the cyclopeptides have highly unusual structures and potent biological activities. However, the majority of the cyclopeptides have not been studied for their biosynthetic mechanisms. In this study, we have established a culture system for the biosynthetic study of heterophyllin B (HB), a cyclopeptide produced by the medicinal plant Pseudostellaria heterophylla. We first developed a shoot culture of P. heterophylla that produced HB consistently under laboratory conditions. Using 14 C-labeled proline as tracer, we showed that labeled HB was produced by the cultured shoots, indicating that this system has de novo biosynthetic activity. Next, we chemically synthesized HB's linear peptide precursor (LHB) and the N-acetyl cysteamine thioester of LHB (LHB-SNAC). When LHB-SNAC was incubated with total cell free extracts of the cultured shoots, a small amount of cyclized product (HB), in addition to the hydrolyzed product (LHB), was produced. The in vivo and in vitro results demonstrate the presence of an HB biosynthetic system, which provides insight into the molecular mechanism for plant cyclopeptide biosynthesis.

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“…Regarding the single mutated trypsin variants, absolutely no synthesis activity could be detected for trypsinG193P, which surprisingly catalyzes only the hydrolysis of the acyl donor ester without any remaining aminolysis. Keeping into account that proline in this position is essential for the efficient peptide bond synthesis activity of native thioesterases, while its exchange with glycine leads to an exclusive hydrolysis activity , more or less the opposite seems to be true for trypsin. In fact, the mutation of glycine with proline in position 193 converts trypsin to an efficient hydrolase lacking any ability of the native parent enzyme for synthesizing peptide bonds.…”

Section: Resultsmentioning

confidence: 99%

“…The latter represents the down‐stream enzymatic activity of nonribosomal peptide synthetases bearing highly sequence specific but excellent peptide bond formation abilities. Interestingly, exchange of this proline moiety with glycine in the surfactin thioesterase A comes along with the loss of aminolysis activity, leading to an enzyme variant with exclusive hydrolysis activity .…”

Section: Introductionmentioning

confidence: 99%

Engineering the oxyanion hole of trypsin for promoting the reverse of proteolysis

Franke

Liebscher

Bordusa

2013

Journal of Peptide Science

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Although proteases are capable of synthesizing peptide bonds via the reverse of proteolysis, they are not proficient at peptide fragment ligation. Further manipulations are needed to shift the native enzyme activity from the cleavage to the synthesis of peptides especially when longer peptides or even proteins are the target molecules of the reaction. This account reports on the synthetic potential of trypsin variants with engineered oxyanion holes mutated by proline mutations, which were designed to minimize proteolytic side reactions during peptide bond synthesis. From the six single and double proline-mutated trypsins, in particular, trypsinQ192P came out as the most promising biocatalyst enabling not only the ligation of cleavage-sensitive peptide fragments but also the selective N-terminal modification of a real protein substrate.

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Solvent Engineering Substantially Enhances the Chemoenzymatic Production of Surfactin

Cited by 12 publications

References 45 publications

The Thioesterase Domain of the Fengycin Biosynthesis Cluster: A Structural Base for the Macrocyclization of a Non-ribosomal Lipopeptide

The Thioesterase Domain of the Fengycin Biosynthesis Cluster: A Structural Base for the Macrocyclization of a Non-ribosomal Lipopeptide

An in vitro system to study cyclopeptide heterophyllin B biosynthesis in the medicinal plant Pseudostellaria heterophylla

Engineering the oxyanion hole of trypsin for promoting the reverse of proteolysis

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