Identification, Characterization, and Three-Dimensional Structure of the Novel Circular Bacteriocin, Enterocin NKR-5-3B, from <i>Enterococcus faecium</i>

Himeno, Kunio; Rosengren, K. Johan; Inoue, Tatsuo; Pérez, Rodney; Colgrave, Michelle L.; Lee, Han Siean; Chan, Lai Yue; Henriques, Sónia Troeira; Fujita, Koji; Ishibashi, Naoki; Zendo, Takeshi; Wilaipun, Pongtep; Nakayama, Jiro; Leelawatcharamas, Vichien; Jikuya, Hiroyuki; Craik, David J.; Sonomoto, Kenji

doi:10.1021/acs.biochem.5b00196

Cited by 61 publications

(77 citation statements)

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“…To determine the minimum inhibitory concentrations (MICs) of the Ent53B Leu1 derivatives, the bacteriocins were purified from their respective phenotypes following our previously reported purification method of NKR-5-3 enterocins [22,23] and were subsequently quantified using the BCA Protein Assay kit (Thermo Scientific). The MICs were determined using the spot-on-lawn assay, as described previously [21,22], using serial dilutions of the purified bacteriocins of known concentrations against a set of indicator bacteria.…”

Section: Rna Isolation and Semi-quantitative Reverse Transcription Pcmentioning

confidence: 99%

“…The MICs were determined using the spot-on-lawn assay, as described previously [21,22], using serial dilutions of the purified bacteriocins of known concentrations against a set of indicator bacteria. MICs were defined as the minimum concentrations that yielded clear zones of growth inhibition on the indicator lawns.…”

Section: Rna Isolation and Semi-quantitative Reverse Transcription Pcmentioning

confidence: 99%

“…Enterocin NKR-5-3B (Ent53B) is a 64-amino acid novel circular bacteriocin produced by Enterococcus faecium NKR-5-3 [21][22][23][24]. The Ent53B structural gene, enkB, encodes an 87-amino acid prepeptide, which consists of a 23-amino acid leader peptide and a 64-amino acid propeptide domain.…”

Section: Introductionmentioning

confidence: 99%

“…The Ent53B structural gene, enkB, encodes an 87-amino acid prepeptide, which consists of a 23-amino acid leader peptide and a 64-amino acid propeptide domain. The head-to-tail circularization of Ent53B occurs between the Leu1 and Trp64 [22]. We recently reported that the maturation of Ent53B from its precursor peptide requires cooperative functioning of four gene products, EnkB1234, that form an enzyme complex [21].…”

Section: Introductionmentioning

confidence: 99%

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Mutations near the cleavage site of enterocin NKR-5-3B prepeptide reveal new insights into its biosynthesis

et al. 2017

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Enterocin NKR-5-3B (Ent53B) is a 64-residue novel circular bacteriocin synthesized from an 87-residue prepeptide. Albeit through a still unknown mechanism, the EnkB1234 biosynthetic enzyme complex processes the prepeptide to yield its mature active, circular form. To gain insights into the key region/residue that plays a role in Ent53 maturation, several mutations near the cleavage site on the precursor peptide were generated. The interaction of the precursor peptide and EnkB1234 appeared to be hydrophobic in nature. At the Leu1 position, only mutations with helix structure-promoting hydrophobic residues (Ala, Ile, Val or Phe) were able to yield the mature Ent53B derivative. In this study, we also highlight the possible conformation-stabilizing role of the Ent53B leader peptide on the precursor peptide for its interaction with its biosynthetic enzyme complex. Any truncations of the leader peptide moiety interfered in the processing of the prepeptide. However, when propeptides of other circular bacteriocins (circularin A, leucocyclicin Q or lactocyclicin Q) were cloned at the C-terminus of the leader peptide, EnkB1234 could not process them to yield a mature bacteriocin. Taken together, these findings offer new perspectives in our understanding of the possible molecular mechanism of the biosynthesis of this circular bacteriocin. These new perspectives will help advance our current understanding to eventually elucidate circular bacteriocin biosynthesis. Understanding the biosynthetic mechanism of circular bacteriocins will materialize their application potential.

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Section: Rna Isolation and Semi-quantitative Reverse Transcription Pcmentioning

confidence: 99%

Section: Rna Isolation and Semi-quantitative Reverse Transcription Pcmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Mutations near the cleavage site of enterocin NKR-5-3B prepeptide reveal new insights into its biosynthesis

et al. 2017

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“…Since then, a number of circular bacteriocins from various bacterial species have been isolated: gassericin A (24), subtilosin A (25), circularin A (3), butyrivibriocin AR10 (26), uberolysin (27), carnocyclin A (28), lactocyclicin Q (29), garvicin ML (30), leucocyclicin Q (31), acidocin B (32), and, most recently, enterocin NKR-5-3B (33). Enterocin NKR-5-3B (Ent53B), one of several bacteriocins produced by Enterococcus faecium NKR-5-3 previously isolated from the Thai fermented fish pla-ra (34)(35)(36), is a 64-amino-acid novel circular bacteriocin with a molecular mass of 6,316.4 Da.…”

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confidence: 99%

Functional Analysis of Genes Involved in the Biosynthesis of Enterocin NKR-5-3B, a Novel Circular Bacteriocin

et al. 2016

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A putative biosynthetic gene cluster of the enterocin NKR-5-3B (Ent53B), a novel circular bacteriocin, was analyzed by sequencing the flanking regions around enkB, the Ent53B structural gene, using a fosmid library. A region approximately 9 kb in length was obtained, and the enkB1, enkB2, enkB3, and enkB4 genes, encoding putative biosynthetic proteins involved in the production, maturation, and secretion of Ent53B, were identified. We also determined the identity of proteins mediating self-immunity against the effects of Ent53B. Heterologous expression systems in various heterologous hosts, such as Enterococcus faecalis and Lactococcus lactis strains, were successfully established. The production and secretion of the mature Ent53B required the cooperative functions of five genes. Ent53B was produced only by those heterologous hosts that expressed protein products of the enkB, enkB1, enkB2, enkB3, and enkB4 genes. Moreover, self-immunity against the antimicrobial action of Ent53B was conferred by at least two independent mechanisms. Heterologous hosts harboring the intact enkB4 gene and/or a combination of intact enkB1 and enkB3 genes were immune to the inhibitory action of Ent53B. IMPORTANCEIn addition to their potential application as food preservatives, circular bacteriocins are now considered possible alternatives to therapeutic antibiotics due to the exceptional stability conferred by their circular structure. The successful practical application of circular bacteriocins will become possible only if the molecular details of their biosynthesis are fully understood. The results of the present study offer a new perspective on the possible mechanism of circular bacteriocin biosynthesis. In addition, since some enterococcal strains are associated with pathogenicity, virulence, and drug resistance, the establishment of the first multigenus host heterologous production of Ent53B has very high practical significance, as it widens the scope of possible Ent53B applications. Bacteriocins are ribosomally synthesized antimicrobial peptides that generally exert their antagonistic activity toward strains that are closely related to the producer strain (1, 2), although an increasing number of bacteriocins have been reported to have a broad activity range (3, 4). In the last decade, the interest in bacteriocins, especially those from lactic acid bacteria (LAB), has increased considerably, as they potentially can be used as natural food preservatives and therapeutic antibiotics (5-8).Over the years, various classification schemes of bacteriocins from Gram-positive bacteria have been suggested, which commonly divide bacteriocins into two groups: class I (lantibiotics) and class II (nonlantibiotics) (9-12). Lantibiotics are small heatstable peptides which contain unusual amino acids, such as lanthionine and/or methyllanthionine, as a result of posttranslational modifications of some common amino acid residues (13,14). In contrast, class II bacteriocins, which are also heat-stable peptides, do not undergo extensive postt...

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A Practical Guide to Structural Aspects of Macrocycles (NMR, X‐Ray, and Modeling)

Craik

Kaas

Wang

2017

Practical Medicinal Chemistry With Macrocycles

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Identification, Characterization, and Three-Dimensional Structure of the Novel Circular Bacteriocin, Enterocin NKR-5-3B, from Enterococcus faecium

Cited by 61 publications

References 51 publications

Mutations near the cleavage site of enterocin NKR-5-3B prepeptide reveal new insights into its biosynthesis

Mutations near the cleavage site of enterocin NKR-5-3B prepeptide reveal new insights into its biosynthesis

Functional Analysis of Genes Involved in the Biosynthesis of Enterocin NKR-5-3B, a Novel Circular Bacteriocin

A Practical Guide to Structural Aspects of Macrocycles (NMR, X‐Ray, and Modeling)

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