Membrane-associated echinocandin B deacylase of Actinoplanes utahensis : purification, characterization, heterologous cloning and enzymatic deacylation reaction

Kreuzman, A. J.; Hodges, Roland; Swartling, James R.; Pohl, Tonya; Ghag, Sandhya; Baker, Patrick J.; McGilvray, D. I.; Yeh, Wu-Kuang

doi:10.1038/sj.jim.2900796

Cited by 24 publications

(18 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Deacylation by Actinoplanes utahensis is attributed to a heterodimeric protein with an exceptionally broad substrate specificity that includes the cyclic hexapeptide antifungal echinocandin B, the cyclic antifungal pseudomycin, and the glycopeptide antibiotic teicoplanin (27). Sequence analysis revealed a number of candidate deacylases within the genomes of actinomycetes, potentially accounting for the deacylation observed in the library.…”

Section: Discussionmentioning

confidence: 99%

Inactivation of the Lipopeptide Antibiotic Daptomycin by Hydrolytic Mechanisms

D’Costa

Mukhtar

Patel

et al. 2012

Antimicrob Agents Chemother

View full text Add to dashboard Cite

The lipopeptide daptomycin is a member of the newest FDA-approved antimicrobial class, exhibiting potency against a broad range of Gram-positive pathogens with only rare incidences of clinical resistance. Environmental bacteria harbor an abundance of resistance determinants orthologous to those in pathogens and thus may serve as an early-warning system for future clinical emergence. A collection of morphologically diverse environmental actinomycetes demonstrating unprecedented frequencies of daptomycin resistance and high levels of resistance by antibiotic inactivation was characterized to elucidate modes of drug inactivation. In vivo studies revealed that hydrolysis plays a key role, resulting in one or both of the following structural modifications: ring hydrolysis resulting in linearization (in 44% of inactivating isolates) or deacylation of the lipid tail (29%). Characterization of the mechanism in actinomycete WAC4713 (a Streptomyces sp. with an MIC of 512 g/ml) demonstrated a constitutive resistance phenotype and established daptomycin's circularizing ester linkage to be the site of hydrolysis. Characterization of the hydrolase responsible revealed it to be likely a serine protease. These studies suggested that daptomycin is susceptible to general proteolytic hydrolysis, which was further supported by studies using proteases of diverse origin. These findings represent the first comprehensive characterization of daptomycin inactivation in any bacterial class and may not only presage a future mechanism of clinical resistance but also suggest strategies for the development of new lipopeptides.T he escalating incidences of infections caused by multidrugresistant bacteria (29, 40) have resulted in an urgent need for the development of novel antimicrobial therapies. In response to this call for new drugs, the lipopeptide daptomycin was approved by the FDA in 2003. This drug represents the first and (so far) only antibiotic of its class to be clinically implemented. Daptomycin exhibits potent and rapid bactericidal activity against an exceptionally broad range of Gram-positive bacteria, including multidrug-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) (4, 12).Although daptomycin's mechanism of action has not fully been elucidated, it is known to act by insertion into the bacterial cell membrane in a Ca 2ϩ -dependent manner, resulting in rapid membrane depolarization (38). Daptomycin is synthesized by the actinomycete Streptomyces roseosporus. Its structure is comprised of 13 amino acids cyclized through an ester linkage between the secondary hydroxyl group of an internal threonine and the carboxyl of the C-terminal kynurenine (Kyn) residues (Fig. 1B). The N-terminal Trp is capped with a C-10 fatty acyl tail. These structural components have been demonstrated to be essential for antimicrobial activity (6, 31). Incidences of decreased susceptibility to daptomycin in clinical settings have been rare thus far, and mechanisms of resistance in ...

show abstract

Section: Discussionmentioning

confidence: 99%

Inactivation of the Lipopeptide Antibiotic Daptomycin by Hydrolytic Mechanisms

D’Costa

Mukhtar

Patel

et al. 2012

Antimicrob Agents Chemother

View full text Add to dashboard Cite

show abstract

“…The three major components, A21978C 1-3 , have 11-, 12-or 13-carbon branched-chain fatty acids, respectively, attached to the terminal amino group of L-Trp (Debono et al, 1987). The fatty acid side chains of A21978C are readily removed by incubation with Actinoplanes utahensis (Boeck et al, 1988) or with the A. utahensis deacylase enzyme produced by a recombinant strain of Streptomyces lividans (Kreuzman et al, 2000), and the cyclic peptide can be reacylated with n-decanoyl fatty acid to produce daptomycin.…”

Section: Introductionmentioning

confidence: 99%

Daptomycin biosynthesis in Streptomyces roseosporus: cloning and analysis of the gene cluster and revision of peptide stereochemistry

et al. 2005

View full text Add to dashboard Cite

Daptomycin is a 13 amino acid, cyclic lipopeptide produced by a non-ribosomal peptide synthetase (NRPS) mechanism in Streptomyces roseosporus. A 128 kb region of S. roseosporus DNA was cloned and verified by heterologous expression in Streptomyces lividans to contain the daptomycin biosynthetic gene cluster (dpt). The cloned region was completely sequenced and three genes (dptA, dptBC, dptD) encoding the three subunits of an NRPS were identified. The catalytic domains in the subunits, predicted to couple five, six or two amino acids, respectively, included a novel activation domain and amino-acid-binding pocket for incorporating the unusual amino acid L-kynurenine (Kyn), three types of condensation domains and an extra epimerase domain (E-domain) in the second module. Novel genes (dptE, dptF ) whose products likely work in conjunction with a unique condensation domain to acylate the first amino acid, as well as other genes (dptI, dptJ) probably involved in supply of the non-proteinogenic amino acids L-3-methylglutamic acid and Kyn, were located next to the NRPS genes. The unexpected E-domain suggested that daptomycin would have D-Asn, rather than L-Asn, as originally assigned, and this was confirmed by comparing stereospecific synthetic peptides and the natural product both chemically and microbiologically.

show abstract

“…Remarkably, although this acylase shares sequence similarity with ␤-lactam acylases (8,11), it has not been reported to show penicillin acylase activity. Preliminary analyses carried out in our laboratory showed that AuAAC was very similar to the ␤-lactam acylase from Streptomyces lavendulae (AY611030), which shows a preference for penicillins with long hydrophobic acyl moieties (21).…”

mentioning

confidence: 99%