ATP Binding in Peptide Synthetases: Determination of Contact Sites of the Adenine Moiety by Photoaffinity Labeling of Tyrocidine Synthetase 1 with 2-Azidoadenosine Triphosphate

Pavela-Vrančić, Maja; Pfeifer, Eva; Liempt, Henk van; Schaefer, Hans-Jochen; Doehren, Hans von; Kleinkauf, Horst

doi:10.1021/bi00186a030

Cited by 45 publications

(22 citation statements)

References 53 publications

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“…The lysine residue of the PPS I motif (NGK), which has been shown to be involved in ATP binding (28), is not conserved in the SnbA family, but several other lysines (512, 530, 533, and 534 as numbered in the alignment) are conserved in the neighboring region. Core sequence H, thought to be involved in adenine binding (29), is not found in the EntE subfamily.…”

Section: Resultsmentioning

confidence: 98%

Pristinamycin I biosynthesis in Streptomyces pristinaespiralis: molecular characterization of the first two structural peptide synthetase genes

Crécy‐Lagard¹,

Blanc²,

Gil³

et al. 1997

J Bacteriol

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Two genes involved in the biosynthesis of the depsipeptide antibiotics pristinamycins I (PI) produced by Streptomyces pristinaespiralis were cloned and sequenced. The 1.7-kb snbA gene encodes a 3-hydroxypicolinic acid:AMP ligase, and the 7.7-kb snbC gene encodes PI synthetase 2, responsible for incorporating L-threonine and L-aminobutyric acid in the PI macrocycle. snbA and snbC, which encode the two first structural enzymes of PI synthesis, are not contiguous. Both genes are located in PI-specific transcriptional units, as disruption of one gene or the other led to PI-deficient strains producing normal levels of the polyunsaturated macrolactone antibiotic pristinamycin II, also produced by S. pristinaespiralis. Analysis of the deduced amino acid sequences showed that the SnbA protein is a member of the adenylate-forming enzyme superfamily and that the SnbC protein contains two amino acid-incorporating modules and a C-terminal epimerization domain. A model for the initiation of PI synthesis analogous to the established model of initiation of fatty acid synthesis is proposed.Pristinamycins I (PI), cyclohexadepsipeptide antibiotics produced by Streptomyces pristinaespiralis, are members of the streptogramin B group. They are coproduced with the polyunsaturated macrolactone antibiotics pristinamycins II (PII), members of the streptogramin A group (6, 52). Like many other small metabolites with a peptide structure, PI are synthesized nonribosomally by large multifunctional enzymes (49). More than 25 years ago, Lipmann emphasized the analogy between nonribosomal peptide synthesis and fatty acid synthesis (25). In his model, large multifunctional enzymes called peptide synthetases (PPSs) catalyze elongation of a peptide covalently linked to a phosphopantetheinyl arm by a thioester bond (for a review, see reference 23). The original version of the thiotemplate multienzymatic mechanism was revised recently, and a multiple-carrier model was proposed (39,43,46,53). According to this model, each amino acid is activated as an aminoacyl adenylate and linked to the enzyme as a thioester with a phosphopantetheinyl group. Elongation then occurs by transfer of the activated carboxyl to the amino group of the next amino acid, thus effecting N-to-C stepwise condensation. Primary structure analysis of several PPS genes resulted in the identification of approximately 1,000-aminoacid (aa)-long modules selectively catalyzing activation and condensation of one amino or hydroxy acid (30, 51). The modules are organized such that they are colinear with the sequence of the oligopeptide. Each module can be subdivided in domains. The activation domain (500 aa) belongs to the large family of adenylate-forming enzymes that includes firefly luciferase and acyl coenzyme A (acyl-CoA) synthetases and contains nine core sequences (named boxes A to I by Pfeifer et al.

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Section: Resultsmentioning

confidence: 98%

Pristinamycin I biosynthesis in Streptomyces pristinaespiralis: molecular characterization of the first two structural peptide synthetase genes

Crécy‐Lagard¹,

Blanc²,

Gil³

et al. 1997

J Bacteriol

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“…The C domain is responsible for peptide bond formation in nonribosomal peptide biosynthesis. (14,41,42,56). Analysis of other peptide synthetases has indicated that each activation domain is responsible for activation of a single amino acid.…”

Section: Resultsmentioning

confidence: 99%

Membrane Localization of Motility, Signaling, and Polyketide Synthetase Proteins in Myxococcus xanthus

2003

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Myxococcus xanthus cells coordinate cellular motility, biofilm formation, and development through the use of cell signaling pathways. In an effort to understand the mechanisms underlying these processes, the inner membrane (IM) and outer membrane (OM) of strain DK1622 were fractionated to examine protein localization. Membranes were enriched from spheroplasts of vegetative cells and then separated into three peaks on a three-step sucrose gradient. The high-density fraction corresponded to the putative IM, the medium-density fraction corresponded to a putative hybrid membrane (HM), and the low-density fraction corresponded to the putative OM. Each fraction was subjected to further separation on discontinuous sucrose gradients, which resulted in discrete protein peaks for each major fraction. The purity and origin of each peak were assessed by using succinate dehydrogenase (SDH) activity as the IM marker and reactivities to lipopolysaccharide core and O-antigen monoclonal antibodies as the OM markers. As previously reported, the OM markers localized to the low-density membrane fractions, while SDH localized to high-density fractions. Immunoblotting was used to localize important motility and signaling proteins within the protein peaks. CsgA, the C-signalproducing protein, and FibA, a fibril-associated protease, were localized in the IM (density, 1.17 to 1.24 g cm ؊3 ). Tgl and Cgl lipoproteins were localized in the OM, which contained areas of high buoyant density (1.21 to 1.24 g cm ؊3 ) and low buoyant density (1.169 to 1.171 g cm ؊3 ). FrzCD, a methyl-accepting chemotaxis protein, was predominantly located in the IM, although smaller amounts were found in the OM. The HM peaks showed twofold enrichment for the type IV pilin protein PilA, suggesting that this fraction contained cell poles. Two-dimensional polyacrylamide gel electrophoresis revealed the presence of proteins that were unique to the IM and OM. Characterization of proteins in an unusually low-density membrane peak (1.072 to 1.094 g cm ؊3 ) showed the presence of Ta-1 polyketide synthetase, which synthesizes the antibiotic myxovirescin A.

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“…Specific activities were calculated from luciferase protein values determined by Western blot analysis and are reported relative to the value obtained at pH 7.8 for each protein: WT (E), L194F/M196L (Ç), S198T (ϫ), L194F/S198T (ϩ), L194F/N197Y/S198T (ࡗ), and L194Y/M196L/S198T/S201T (ᮍ). (16), but now that the luciferase structure is available (10), it can readily be seen that sites of photoaffinity labeling are spatially close to 198 -207 even though they are distant in the primary structure. This region of the protein likely constitutes a portion of the ATP binding domain.…”

Section: Resultsmentioning

confidence: 99%

Mutation of a Protease-sensitive Region in Firefly Luciferase Alters Light Emission Properties

Thompson¹,

Geoghegan²,

Lloyd³

et al. 1997

Journal of Biological Chemistry

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Luciferase (EC 1.13.12.7) from the North American firefly, Photinus pyralis, is widely used as a reporter enzyme in cell biology. One of its distinctive properties is a pronounced susceptibility to proteolytic degradation that causes luciferase to have a very short intracellular half-life. To define the structural basis for this behavior and possibly facilitate the design of more stable forms of luciferase, limited proteolysis studies were undertaken using trypsin and chymotrypsin to identify regions of the protein whose accessible and flexible character rendered them especially sensitive to cleavage. Regions of amino acid sequence 206 -220 and 329 -341 were found to be sensitive, and because the region around 206 -220 had high homology with other luciferases, CoA ligases, and peptidyl synthetases, this region was selected for mutagenesis experiments intended to determine which of its amino acids were essential for activity. Surprisingly, many highly conserved residues including Ser 198 , Ser 201 , Thr 202 , and Gly 203 could be mutated with little effect on the luminescent activity of P. pyralis luciferase. One mutation, however, S198T, caused several alterations in enzymatic properties including shifting the pH optimum from 8.1 to 8.7, lowering the K m for Mg-ATP by a factor of 4 and increasing the half-time for light emission decay by a factor of up to 150. While the S198T luciferase was less active than wild type, activity could be restored by the introduction of the additional L194F and N197Y mutations. In addition to indicating the involvement of this region in ATP binding, these results provide a new form of the enzyme that affords a more versatile reporter system. Luciferase (EC 1.13.12.7) from the North American firefly, Photinus pyralis, is widely used as a reporter enzyme in studies of gene expression (1). Firefly luciferase first catalyzes the formation of a bound luciferin-AMP, which then reacts with O 2 to generate oxyluciferin-AMP in an excited state. Breakdown of this intermediate to oxyluciferin, CO 2 , and AMP is accompanied by the emission of a photon. The high quantum yield for this process, nearly a single photon emitted per reacted luciferin molecule (2), reflects not only an efficient catalytic machinery but also a highly favorable environment for radiative decay of an excited state.The kinetics of light emission can be modulated by coenzyme A, but this is not a required substrate (3, 4). Luciferase can also function as a ligase (5) as the AMP group of the enzyme⅐luciferyl-AMP complex can be transferred to ATP to produce diadenosine tetraphosphate.Despite many studies, relatively little was known about the structure of luciferase or the nature of its active site until recently. Cloning and sequencing of luciferase from the firefly (6) and its homologues from several beetles (reviewed in Ref . 7) have shown that these enzymes are related to certain acyltransferases (8, 9). Alignment of these related sequences reveals conserved residues that may be important for enzymatic activity. For exam...

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ATP Binding in Peptide Synthetases: Determination of Contact Sites of the Adenine Moiety by Photoaffinity Labeling of Tyrocidine Synthetase 1 with 2-Azidoadenosine Triphosphate

Cited by 45 publications

References 53 publications

Pristinamycin I biosynthesis in Streptomyces pristinaespiralis: molecular characterization of the first two structural peptide synthetase genes

Pristinamycin I biosynthesis in Streptomyces pristinaespiralis: molecular characterization of the first two structural peptide synthetase genes

Membrane Localization of Motility, Signaling, and Polyketide Synthetase Proteins in Myxococcus xanthus

Mutation of a Protease-sensitive Region in Firefly Luciferase Alters Light Emission Properties

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