Enzymatical and microbial degradation of cyclic dipeptides (diketopiperazines)

AMB Express, Aug 2013

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.

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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 - 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)


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Mareike Perzborn, Christoph Syldatk, Jens Rudat. Enzymatical and microbial degradation of cyclic dipeptides (diketopiperazines), AMB Express, 2013, pp. 51, Volume 3, Issue 1, DOI: 10.1186/2191-0855-3-51