Bacilysin biosynthesis by a partially-purified enzyme fraction from Bacillus subtilis

Yazgan, Ayten; Özcengiz, Gülay; Özcengīz, Erkan; Kılınç, Kamer; Marahiel, Mohamed A.; Alaeddinoğlu, N. Gürdal

doi:10.1016/s0141-0229(01)00401-x

Cited by 18 publications

(10 citation statements)

References 28 publications

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“…Since an enzyme that belongs to the ATP-dependent carboxylate-amine/thiol ligase superfamily transfers phosphate from ATP to its acyl substrate, it forms ADP and phosphate as reaction products (9,14), whereas aminoacyl-tRNA A previous report has suggested that the bacilysin-synthesizing reaction was carried out in an NRPS-like manner (29). The discrepancy with our results may arise from a difference in enzyme purity; the former study used a partially purified fraction whereas we used a fraction purified to apparent homogeneity (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…This means the formation of a futile cycle between YwfE protein and peptidases that is meaningless for the cells except in some specific circumstances in which ATP is in excess. Second, the expression of ywfE is strictly controlled and coordinated with that of the bacilysin biosynthetic gene cluster (15,29). Third, a ywfE-deficient mutant was reported to grow normally (11,28).…”

Section: Discussionmentioning

confidence: 99%

“…The protein is encoded by the gene cluster though the exact function of each gene has not been elucidated (12,18,29). Confirmation of YwfE protein activity.…”

Section: ϫ4mentioning

confidence: 99%

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ywfEinBacillus subtilisCodes for a Novel Enzyme,l-Amino Acid Ligase

Tabata

Ikeda²,

Hashimoto³

2005

J Bacteriol

113

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The ATP-dependent carboxylate-amine/thiol ligase superfamily is known to contain enzymes catalyzing the formation of various types of peptide, such as D-alanyl-D-alanine, polyglutamate, and ␥-peptide, but, curiously, no enzyme synthesizing ␣-dipeptides of L-amino acids is known. We attempted to find such an enzyme. By in silico screening based on the consensus sequence of the superfamily followed by an in vitro assay with purified enzyme to avoid the degradation of the peptide(s) synthesized, ywfE of Bacillus subtilis was found to code for the activity forming L-alanyl-L-glutamine from L-alanine and L-glutamine with hydrolysis of ATP to ADP. No AMP was formed, supporting the idea that the enzyme belongs to the superfamily. Surprisingly, the enzyme accepted a wide variety of L-amino acids. Among 231 combinations of L-amino acids tested, reaction products were obtained for 111 combinations and 44 kinds of ␣-dipeptides were confirmed by high-performance liquid chromatography analyses, while no tripeptide or longer peptide was detected and the D-amino acids were inert. From these results, we propose that ywfE encodes a new member of the superfamily, L-amino acid ligase.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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ywfEinBacillus subtilisCodes for a Novel Enzyme,l-Amino Acid Ligase

Tabata

Ikeda²,

Hashimoto³

2005

J Bacteriol

113

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“…Bacilysin was also determined by thin-layer chromatography (TLC) followed by bioautography (Yazgan et al 2001b;Yazgan-Karataş et al 2003). Culture supernatants of strains were extracted three times with one-fourth column of butanol by vortexing for 20 s followed by 2 min centrifugation at 13,000 rpm to separate the organic phase.…”

Section: Extraction Of Culture Fluids and Thin Layer Chromatographymentioning

confidence: 99%

The novel gene yvfI in Bacillus subtilis is essential for bacilysin biosynthesis

Köroğlu

Kurt-Gür

Unlü

et al. 2008

Antonie van Leeuwenhoek

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Using transposon mutagenesis in Bacillus subtilis PY79, three independent mutants defective in production of bacilysin were isolated. To identify the genes in these mutant loci affecting bacilysin biosynthesis, the inserted transposon and its flanking regions were cloned and sequenced from each mutant. Transposon insertions in these three mutants were found to be in the yvfI gene which encodes an unknown protein similar to GntR family transcriptional regulators. For further confirmation, deletion mutants were constructed in which nucleotides 196-314 of the yvfI gene were removed. All resulting yvfI (Delta196-314)::spc deletion mutants exhibited bacilysin-negative phenotypes, as in the case of the yvfI::Tn10::spc insertional mutants. The lacR gene, encoding a transcriptional regulator, resides immediately downstream from the yvfI gene. Therefore, an insertion mutation was created in the lacR gene to demonstrate that the bacilysin negative phenotype is actually due to the mutation in the yvfI gene and not a polar effect of yvfI mutation on the downstream gene. As expected, all resulting lacR mutant derivatives of PY79 still produced bacilysin.

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“…Our group reported partial purification of a biosynthetic enzyme fraction (ca. 125 kDa) by fast flow gel permeation followed by anion exchange FPLC [29]. Enzyme characterization studies, overall, showed that bacilysin biosynthesis does not fit to the general mechanisms of NRPSs.…”

Section: Biochemistry and Genetics Of Bacilysin Biosynthesismentioning

confidence: 96%