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Adenylation Domain (adenylation + domain)

Distribution by Scientific Domains
Chemistry100%

Selected Abstracts

Mapping the Limits of Substrate Specificity of the Adenylation Domain of TycA

CHEMBIOCHEM, Issue 4 2009
Benoit R. M. Villiers
Abstract The limits and potential of substrate promiscuity of the adenylation domain of tyrocidine synthetase 1 were systematically explored. Substrate acceptance is governed by hydrophobic effects (as shown by the correlation of kcat/KM and side-chain log,P), shape complementarity and steric exclusion. The quantification of these factors provides ground rules for understanding and possibly evolving substrate specificity in this class of enzymes. The catalytic potential of tyrocidine synthetase 1 (TycA) was probed by the kinetic characterization of its adenylation activity. We observed reactions with 30 substrates, thus suggesting some substrate promiscuity. However, although the TycA adenylation (A) domain was able to accommodate alternative substrates, their kcat/KM values ranged over six orders of magnitude. A comparison of the activities allowed the systematic mapping of the substrate specificity determinants of the TycA A-domain. Hydrophobicity plays a major role in the recognition of substrate analogues but can be combined with shape complementarity, conferring higher activity, and/or steric exclusion, leading to substantial discrimination against larger substrates. A comparison of the kcat/KM values of the TycA A-domain and phenylalanyl-tRNA synthetase showed that the level of discrimination was comparable in the two enzymes for the adenylation reaction and suggested that TycA was also subjected to high selective pressure. The specificity patterns observed and the quantification of alternative activities provide a basis for exploring possible paths for the future directed evolution of A-domain specificity. [source]

Structure of the adenylation domain of NAD+ -dependent DNA ligase from Staphylococcus aureus

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009
Seungil Han
DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5,-phosphate and 3,-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD+ -dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD+ -dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD+ -binding pocket and the `C2 tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors. [source]

Biosynthesis of the Myxobacterial Antibiotic Corallopyronin A

CHEMBIOCHEM, Issue 9 2010
Özlem Erol
Abstract Corallopyronin A is a myxobacterial compound with potent antibacterial activity. Feeding experiments with labelled precursors resulted in the deduction of all biosynthetic building blocks for corallopyronin A and revealed an unusual feature of this metabolite: its biosynthesis from two chains, one solely PKS-derived and the other NRPS/PKS-derived. The starter molecule is believed to be carbonic acid or its monomethyl ester. The putative corallopyronin A biosynthetic gene cluster is a trans-AT-type mixed PKS/NRPS gene cluster, containing a ,-branching cassette. Striking features of this gene cluster are a NRPS-like adenylation domain that is part of a PKS-type module and is believed to be responsible for glycine incorporation, as well as split modules with individual domains occurring on different genes. It is suggested that CorB is a trans-acting ketosynthase and it is proposed that it catalyses the Claisen condensation responsible for the interconnection of the two chains. Additionally, the stereochemistry of corallopyronin A was deduced by a combination of a modified Mosher's method and ozonolysis with subsequent chiral GC analyses. [source]

Mapping the Limits of Substrate Specificity of the Adenylation Domain of TycA

CHEMBIOCHEM, Issue 4 2009
Benoit R. M. Villiers
Abstract The limits and potential of substrate promiscuity of the adenylation domain of tyrocidine synthetase 1 were systematically explored. Substrate acceptance is governed by hydrophobic effects (as shown by the correlation of kcat/KM and side-chain log,P), shape complementarity and steric exclusion. The quantification of these factors provides ground rules for understanding and possibly evolving substrate specificity in this class of enzymes. The catalytic potential of tyrocidine synthetase 1 (TycA) was probed by the kinetic characterization of its adenylation activity. We observed reactions with 30 substrates, thus suggesting some substrate promiscuity. However, although the TycA adenylation (A) domain was able to accommodate alternative substrates, their kcat/KM values ranged over six orders of magnitude. A comparison of the activities allowed the systematic mapping of the substrate specificity determinants of the TycA A-domain. Hydrophobicity plays a major role in the recognition of substrate analogues but can be combined with shape complementarity, conferring higher activity, and/or steric exclusion, leading to substantial discrimination against larger substrates. A comparison of the kcat/KM values of the TycA A-domain and phenylalanyl-tRNA synthetase showed that the level of discrimination was comparable in the two enzymes for the adenylation reaction and suggested that TycA was also subjected to high selective pressure. The specificity patterns observed and the quantification of alternative activities provide a basis for exploring possible paths for the future directed evolution of A-domain specificity. [source]

Structure of the adenylation domain of NAD+ -dependent DNA ligase from Staphylococcus aureus

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009
Seungil Han
DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5,-phosphate and 3,-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD+ -dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD+ -dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD+ -binding pocket and the `C2 tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors. [source]

Nonribosomal Peptide Synthesis in Schizosaccharomyces pombe and the Architectures of Ferrichrome-Type Siderophore Synthetases in Fungi

CHEMBIOCHEM, Issue 4 2006
Torsten Schwecke Dr.
Abstract A nonribosomal peptide synthetase (NRPS) in Schizosaccharomyces pombe, which possesses an unusual structure incorporating three adenylation domains, six thiolation domains and six condensation domains, has been shown to produce the cyclohexapeptide siderophore ferrichrome. One of the adenylation domains is truncated and contains a distorted key motif. Substrate-binding specificities of the remaining two domains were assigned by molecular modelling to glycine and to N -acetyl- N -hydroxy- L -ornithine. Hexapeptide siderophore synthetase genes of Magnaporthe grisea and Fusarium graminearum were both identified and analyzed with respect to substrate-binding sites, and the predicted product ferricrocin was identified in each. A comparative analysis of these synthetase systems, including those of the basidiomycete Ustilago maydis, the homobasidiomycete Omphalotus olearius and the ascomycetes Aspergillus nidulans, Aspergillus fumigatus, Fusarium graminearum, Cochliobolus heterostrophus, Neurospora crassa and Aureobasidium pullulans, revealed divergent domain compositions with respect to their number and positioning, although all produce similar products by iterative processes. A phylogenetic analysis of both NRPSs and associated L - N5 -ornithine monooxygenases revealed that ferrichrome-type siderophore biosynthesis has coevolved in fungi with varying in trans interactions of NRPS domains. [source]

Structure and Biosynthesis of Myxochromides S1,3 in Stigmatella aurantiaca: Evidence for an Iterative Bacterial Type I Polyketide Synthase and for Module Skipping in Nonribosomal Peptide Biosynthesis,

CHEMBIOCHEM, Issue 2 2005
Silke C. Wenzel Dipl.-Chem.
Abstract The myxobacterium Stigmatella aurantiaca DW4/3,1 harbours an astonishing variety of secondary metabolic gene clusters, at least two of which were found by gene inactivation experiments to be connected to the biosynthesis of previously unknown metabolites. In this study, we elucidate the structures of myxochromides S1,3, novel cyclic pentapeptide natural products possessing unsaturated polyketide side chains, and identify the corresponding biosynthetic gene locus, made up of six nonribosomal peptide synthetase modules. By analyzing the deduced substrate specificities of the adenylation domains, it is shown that module 4 is most probably skipped during the biosynthetic process. The polyketide synthase MchA harbours only one module and is presumably responsible for the formation of the variable complete polyketide side chains. These data indicate that MchA is responsible for an unusual iterative polyketide chain assembly. [source]