Multifunctional Peptide Synthetases

H, von Döhren; Keller, Ullrich; Vater, Joachim; Zocher, Rainer

doi:10.1021/cr9600262

Cited by 234 publications

(162 citation statements)

References 204 publications

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“…[16] In general, each adenylation domain contains an eight-residue, nonlinear sequence motif that is responsible for binding the substrate amino acid. The four adenylation domains of Afpes1 were aligned with the phenylalanine-activating adenylation domain of GrsA [17] to identify each eight-residue motif.…”

Section: Resultsmentioning

confidence: 99%

A 4′‐Phosphopantetheinyl Transferase Mediates Non‐Ribosomal Peptide Synthetase Activation in Aspergillus fumigatus

et al. 2005

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Aspergillus fumigatus is a significant human pathogen. Non‐ribosomal peptide (NRP) synthesis is thought to be responsible for a significant proportion of toxin and siderophore production in the organism. Furthermore, it has been shown that 4′‐phosphopantetheinylation is required for the activation of key enzymes involved in non‐ribosomal peptide synthesis in other species. Here we report the cloning, recombinant expression and functional characterisation of a 4′‐phosphopantetheinyl transferase from A. fumigatus and the identification of an atypical NRP synthetase (Afpes1), spanning 14.3 kb. Phylogenetic analysis has shown that the NRP synthetase exhibits greatest identity to NRP synthetases from Metarhizium anisolpiae (PesA) and Alternaria brassicae (AbrePsy1). Northern hybridisation and RT‐PCR analysis have confirmed that both genes are expressed in A. fumigatus. A 120 kDa fragment of the A. fumigatus NRP synthetase, containing a putative thiolation domain, was cloned and expressed in the baculovirus expression system. Detection of a 4′‐phosphopantetheinylated peptide (SFSAMK) from this protein, by MALDI‐TOF mass spectrometric analysis after coincubation of the 4′‐phosphopantetheinyl transferase with the recombinant NRP synthetase fragment and acetyl CoA, confirms that it is competent to play a role in NRP synthetase activation in A. fumigatus. The 4′‐phosphopantetheinyl transferase also activates, by 4′‐phosphopantetheinylation, recombinant α‐aminoadipate reductase (Lys2p) from Candida albicans, a key enzyme involved in lysine biosynthesis.

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Section: Resultsmentioning

confidence: 99%

A 4′‐Phosphopantetheinyl Transferase Mediates Non‐Ribosomal Peptide Synthetase Activation in Aspergillus fumigatus

et al. 2005

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“…Amino acid alignment of the putative AMP-binding domains of CPS1 and its fungal and animal orthologs confirmed the presence of conserved core sequences (cores 1-5) found in AMP domaincontaining enzymes (20)(21)(22) (Fig. 9).…”

Section: Resultsmentioning

confidence: 55%

“…Although database searches did not reveal other functional domains, manual alignment of amino acids between CPS1A and CPS1B suggested that CPS1 has a thiolation (T, also called peptidyl carrier protein) domain that is also found in all NRPSs 3Ј of each AMP domain (21,22) (Fig. 9).…”

Section: Resultsmentioning

confidence: 99%

A novel class of gene controlling virulence in plant pathogenic ascomycete fungi

Kroken

Lee

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Insertional mutants of the fungal maize pathogen Cochliobolus heterostrophus were screened for altered virulence. One mutant had 60% reduction in lesion size relative to WT but no other detectable change in phenotype. Analysis of sequence at the insertion site revealed a gene (CPS1) encoding a protein with two AMP-binding domains. CPS1 orthologs were detected in all Cochliobolus spp. examined, in several other classes of ascomycete fungi, and in animals but not in basidiomycete fungi, bacteria, or plants. Phylogenetic analysis suggested that CPS1 represents a previously undescribed subset of adenylate-forming enzymes that have diverged from certain acyl-CoA ligases, which in bacteria are involved in biosynthesis of nonribosomal peptides or polyketide͞peptide hybrids. Disruption of CPS1 caused reduced virulence of both race T and race O of C. heterostrophus on maize, of Cochliobolus victoriae on oats, and of Gibberella zeae on wheat. These results suggest that CPS1 functions as a general fungal virulence factor in plant pathogenic ascomycetes.

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“…A wide variety of bioactive linear, cyclic and branched cyclic peptide products are synthesized via the non-ribosomal protein thiotemplate mechanism catalysed by multifunctional peptide synthetases [1,2]. The ribosomal system is restricted to the 20 proteinogenic amino acids, whereas more than 300 derivatives, including hydroxy, N-methylated and -amino acids, are known to be incorporated into these secondary metabolites.…”

Section: Introductionmentioning

confidence: 99%

“…This elevated energy input has been attributed to loss of mis-activated intermediates by hydrolysis in an Abbreviations used : apo-TY1, apo-form of tyrocidine synthetase 1 ; IPP, inorganic pyrophosphatase ; PNP, purine nucleoside phosphorylase ; AMMPR, 2-amino-6-mercapto-7-methylpurine ribonucleoside ; E, enzyme ; PP i , pyrophosphate. 1 To whom correspondence should be addressed (e-mail Doehren!chem.tu-berlin.de).…”

Section: Introductionmentioning

confidence: 99%

Editing of non-cognate aminoacyl adenylates by peptide synthetases

Pavela-Vrančić

Dieckmann²,

Döhren³

et al. 1999

Biochemical Journal

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Non-ribosomally formed peptides display both highly conserved and variable amino acid positions, the variations leading to a wide range of peptide families. Activation of the amino acid substrate proceeds in analogy to the ribosomal biosynthetic mechanism generating aminoacyl adenylate and acyl intermediates. To approach the mechanism of fidelity of amino acid selection, the stability of the aminoacyl adenylates was studied by employing a continuous coupled spectrophotometric assay. The apo-form of tyrocidine synthetase 1 (apo-TY1) was used, generating an -phenylalanyl-adenylate intermediate stabilized by the interaction of two structural subdomains of the adenylation domain. Adenylates of substrate analogues have shown variable and reduced degrees of stability, thus leading to an enhanced generation of pyrophosphate due to hydrolysis and continuous adenylate formation. Discrimination of the non-

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Multifunctional Peptide Synthetases

Cited by 234 publications

References 204 publications

A 4′‐Phosphopantetheinyl Transferase Mediates Non‐Ribosomal Peptide Synthetase Activation in Aspergillus fumigatus

A 4′‐Phosphopantetheinyl Transferase Mediates Non‐Ribosomal Peptide Synthetase Activation in Aspergillus fumigatus

A novel class of gene controlling virulence in plant pathogenic ascomycete fungi

Editing of non-cognate aminoacyl adenylates by peptide synthetases

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