Enzymatical and microbial degradation of cyclic dipeptides (diketopiperazines)
AMB Express
Enzymatical and microbial degradation of cyclic dipeptides (diketopiperazines)
Mareike Perzborn 0
Christoph Syldatk 0
Jens Rudat 0
0 Karlsruhe Institute of Technology, Institute of Process Engineering in Life Sciences, Section II: Technical Biology , Engler-Bunte-Ring 1, 76131 Karlsruhe , Germany
Diketopiperazines (DKPs) are cyclic dipeptides, representing an abundant class of biologically active natural compounds. Despite their widespread occurrence in nature, little is known about their degradation. In this study, the enzymatical and microbial cleavage of DKPs was investigated. Peptidase catalyzed hydrolysis of certain DKPs was formerly reported, but could not be confirmed in this study. While testing additional peptidases and DKPs no degradation was detected, indicating peptidase stability of the peptide bond in cyclic dipeptides. Besides confirmation of the reported degradation of cyclo(L-Asp-L-Phe) by Paenibacillus chibensis (DSM 329) and Streptomyces flavovirens (DSM 40062), cleavage of cyclo(L-Asp-L-Asp) by DSM 329 was detected. Other DKPs were not hydrolyzed by both strains, demonstrating high substrate specificity. The degradation of cyclo(L-Asp-L-Phe) by DSM 40062 was shown to be inducible. Three strains, which are able to hydrolyze hydantoins and dihydropyrimidines, were identified for the degradation of DKPs: Leifsonia sp. K3 (DSM 27212) and Bacillus sp. A16 (DSM 25052) cleaved cyclo(DL-Ala-DL-Ala) and cyclo(L-Gly-L-Phe), and Rhizobium sp. NA04-01 (DSM 24917) degraded cyclo(L-Asp-L-Phe), cyclo(L-Gly-L-Phe) and cyclo(L-Asp-L-Asp). The first enantioselective cleavage of cyclo(DL-Ala-DL-Ala) was detected with the newly isolated strains Paenibacillus sp. 32A (DSM 27214) and Microbacterium sp. 40A (DSM 27211). Cyclo(L-Ala-D-Ala) and cyclo(L-Ala-L-Ala) were completely degraded, whereas the enantiomer cyclo(D-Ala-D-Ala) was not attacked. Altogether, five bacterial strains were newly identified for the cleavage of DKPs. These bacteria may be of value for industrial purposes, such as degradation of undesirable DKPs in food and drugs and production of (enantiopure) dipeptides and amino acids.
Diketopiperazines; Cyclic dipeptides; Peptide bond; Degradation; Hydrolysis; Biotransformation; Cyclic amidases; Peptidases
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Introduction
Diketopiperazines (DKPs) are the smallest possible cyclic
peptides composed of two α-amino acids. They are
abundant natural compounds produced by a variety of
organisms (Borthwick 2012): bacteria like Bacillus
subtilis (Elkahoui et al. 2012) and Streptomyces sp.
(Johnson et al. 1951), fungi, e.g., Alternaria alternata
(Stierle et al. 1988) and Penicillium sp. (Du et al. 2009),
and sponges, for example, Dysidea fragilis (Su et al.
1993). They were identified in mammals, e.g., in rat and
monkey brains, human central nervous system,
gastrointestinal tract and blood (Prasad 1988). Besides the
widespread DKP biosynthesis, they occur as chemical
degradation products in e.g., roasted coffee (Ginz and
Engelhardt 2000), stewed beef (Chen et al. 2009) and
beer (Gautschi et al. 1997). The identified DKPs were
described to cause a bitter taste in these foods.
Furthermore, DKPs are formed as decomposition products of
drugs by cyclization, e.g., of the aminopenicillin
antibiotic amoxicillin (Lamm et al. 2009).
Amoxicillin2,5-diketopiperazine, the chemically stable form of
amoxicillin was detected in wastewater samples, and
may elicit allergic reactions in human consumers of
water and food of animal origin (Lamm et al. 2009).
Identification of DKP cleaving enzymes or
microorganisms could be important for the degradation of
these interfering by-products in food and drug industry.
DKPs exhibit diverse bioactivities including antibacterial
(Fdhila et al. 2003), antifungal (Ström et al. 2002) and
antiviral activity (Sinha et al. 2004), as well as cytotoxicity
(McCleland et al. 2004) and phytotoxicity (Stierle et al.
1988). Moreover, DKPs were shown to act as quorum
sensing molecules (Ryan and Dow 2008).
Cyclo(L-Pro-LTyr), used in this study, was identified in culture
supernatant of e.g., Pseudomonas aeruginosa, and was shown to
activate an N-acylhomoserine lactone biosensor (Holden
et al. 1999).
Little is known about the biodegradation of these
molecules. There are only few studies describing the
enzymatical or microbial hydrolysis of DKPs.
Some peptidases (also termed proteases, EC 3.4.X.X)
are reported to cleave the peptide bonds in DKPs.
However, some of the results are inconsistent and to the best
of our knowledge the last report about this topic was
published in 1940.
The first cleavage of a peptide bond in DKPs was
reported for cyclo(Asp-Asp), cyclo(Gly-Asp) and cyclo
(Gly-Glu) by trypsin (Matsui 1933). However, hydrolysis
of cyclo(Gly-Asp) and cyclo(Gly-Glu) by trypsin was
disproved afterwards (Akabori and Takase 1936).
Cleavage of cyclo(Asp-Asp) and cyclo(Gly-Glu) by papain was
demonstrated by Shibata and Tazawa (1936), but the last
could not be confirmed by Akabori an (...truncated)