References of "Molnar, I"
     in
Bookmark and Share    
Full Text
Peer Reviewed
See detailThe pipecolate-incorporating enzyme for the biosynthesis of the immunosuppressant rapamycin: Nucleotide sequence analysis, disruption and heterologous expression of rapP from Streptomyces hygroscopicus.
König, Ariane UL; Schwecke, T.; Molnár, I. et al

in European Journal of Biochemistry (1997), 247(2), 526-534

An open reading frame (rapP) encoding the putative pipecolate-incorporating enzyme (PIE) has been identified in the gene cluster for the biosynthesis of rapamycin in Streptomyces hygroscopicus. Conserved ... [more ▼]

An open reading frame (rapP) encoding the putative pipecolate-incorporating enzyme (PIE) has been identified in the gene cluster for the biosynthesis of rapamycin in Streptomyces hygroscopicus. Conserved amino acid sequence motifs for ATP binding, ATP hydrolysis, adenylate formation, and 4'-phosphopantetheine attachment were identified by sequence comparison with authentic peptide synthetases. Disruption of rapP by phage insertion abolished rapamycin production in S. hygroscopicus, and the production of the antibiotic was specifically restored upon loss of the inserted phage by a second recombination event. rapP was expressed in both Escherichia coli and Streptomyces coelicolor, and recombinant PIE was purified to homogeneity from both hosts. Although low-level incorporation of [14C]beta-alanine into recombinant PIE isolated from E. coli was detected, formation of the covalent acylenzyme intermediate could only be shown with the PIE from S. coelicolor, suggesting that while the recombinant PIE from S. coelicolor was phosphopantetheinylated, only a minor proportion of the recombinant enzyme from E. coli was post-translationally modified. [less ▲]

Detailed reference viewed: 93 (5 UL)
Full Text
See detailThe nature of the starter unit for the rapamycin polyketide synthase
König, Ariane UL; Schwecke, T.; Molnár, I. et al

in Angewandte Chemie International Edition (1996), 35(19), 2249-2251

A remarkably high level of incorporation is observed when 2,2,5,5-tetradeutero -3,4- dihydroxycyclo -hexanecarboxylic acid is fed to the rapamycin-producing organism. Intriguingly, however, analysis of ... [more ▼]

A remarkably high level of incorporation is observed when 2,2,5,5-tetradeutero -3,4- dihydroxycyclo -hexanecarboxylic acid is fed to the rapamycin-producing organism. Intriguingly, however, analysis of the gene sequence for the rapamycin polyketide synthase has suggested that the free acid may not normally be involved in rapamycin biosynthesis. [less ▲]

Detailed reference viewed: 93 (4 UL)
Full Text
Peer Reviewed
See detailOrganisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of the enzymatic domains in the modular polyketide synthase
Aparicio, J. F.; Molnar, I.; Schwecke, T. et al

in Gene (1996), 169(1), 9-16

The three giant multifunctional polypeptides of the rapamycin (Rp)-producing polyketide synthase (RAPS1, RAPS2 and RAPS3) have recently been shown to contain 14 separate sets, or modules, of enzyme ... [more ▼]

The three giant multifunctional polypeptides of the rapamycin (Rp)-producing polyketide synthase (RAPS1, RAPS2 and RAPS3) have recently been shown to contain 14 separate sets, or modules, of enzyme activities, each module catalysing a specific round of polyketide chain extension. Detailed sequence comparison between these protein modules has allowed further characterisation of aa that may be important in catalysis or specificity. The acyl-carrier protein (ACP), beta-ketoacyl-ACP synthase (KS) and acyltransferase (AT) domains (the core domains) have an extremely high degree of mutual sequence homology. The KS domains in particular are almost perfect repeats over their entire length. Module 14 shows the least homology and is unique in possessing only core domains. The enoyl reductase (ER), beta-ketoacyl-ACP reductase (KR) and dehydratase (DH) domains are present even in certain modules where they are not apparently required. Four DH domains can be recognised as inactive by characteristic deletions in active site sequences, but for two others, and for KR and ER in module 3, the sequence is not distinguishable from that of active counterparts in other modules. The N terminus of RAPS1 contains a novel coenzyme A ligase (CL) domain that activates and attaches the shikimate-derived starter unit, and an ER activity that may modify the starter unit after attachment. The sequence comparison has revealed the surprisingly high sequence similarity between inter-domain 'linker' regions, and also a potential amphipathic helix at the N terminus of each multienzyme subunit which may promote dimerisation into active species. [less ▲]

Detailed reference viewed: 74 (2 UL)
Full Text
Peer Reviewed
See detailOrganisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of the genes flanking the polyketide synthase
Aparicio, J. F.; Molnár, I.; Schwecke, T. et al

in Gene (1996), 169(1), 1-7

The three giant multifunctional polypeptides of the rapamycin (Rp)-producing polyketide synthase (RAPS1, RAPS2 and RAPS3) have recently been shown to contain 14 separate sets, or modules, of enzyme ... [more ▼]

The three giant multifunctional polypeptides of the rapamycin (Rp)-producing polyketide synthase (RAPS1, RAPS2 and RAPS3) have recently been shown to contain 14 separate sets, or modules, of enzyme activities, each module catalysing a specific round of polyketide chain extension. Detailed sequence comparison between these protein modules has allowed further characterisation of aa that may be important in catalysis or specificity. The acyl-carrier protein (ACP), beta-ketoacyl-ACP synthase (KS) and acyltransferase (AT) domains (the core domains) have an extremely high degree of mutual sequence homology. The KS domains in particular are almost perfect repeats over their entire length. Module 14 shows the least homology and is unique in possessing only core domains. The enoyl reductase (ER), beta-ketoacyl-ACP reductase (KR) and dehydratase (DH) domains are present even in certain modules where they are not apparently required. Four DH domains can be recognised as inactive by characteristic deletions in active site sequences, but for two others, and for KR and ER in module 3, the sequence is not distinguishable from that of active counterparts in other modules. The N terminus of RAPS1 contains a novel coenzyme A ligase (CL) domain that activates and attaches the shikimate-derived starter unit, and an ER activity that may modify the starter unit after attachment. The sequence comparison has revealed the surprisingly high sequence similarity between inter-domain 'linker' regions, and also a potential amphipathic helix at the N terminus of each multienzyme subunit which may promote dimerisation into active species. [less ▲]

Detailed reference viewed: 125 (2 UL)
Full Text
See detailDivergent sequence motifs correlated with the substrate specificity of (methyl)malonyl-CoA:acyl carrier protein transacylase domains in modular polyketide synthases.
Haydock, S. F.; Aparicio, J. F.; König, Ariane UL et al

in FEBS Letters (1995), 374(2), 246-248

The amino acid sequences of a large number of polyketide synthase domains that catalyse the transacylation of either methylmalonyl-CoA or malonyl-CoA onto acyl carrier protein (ACP) have been compared ... [more ▼]

The amino acid sequences of a large number of polyketide synthase domains that catalyse the transacylation of either methylmalonyl-CoA or malonyl-CoA onto acyl carrier protein (ACP) have been compared. Regions were identified in which the acyltransferase sequences diverged according to whether they were specific for malonyl-CoA or methylmalonyl-CoA. These differences are sufficiently clear to allow unambiguous assignment of newly-sequenced acyltransferase domains in modular polyketide synthases. Comparison with the recently-determined structure of the malonyltransferase from Escherichia coli fatty acid synthase showed that the divergent region thus identified lies near the acyltransferase active site, though not close enough to make direct contact with bound substrate. [less ▲]

Detailed reference viewed: 98 (6 UL)
Full Text
Peer Reviewed
See detailThe biosynthetic cluster for the polyketide immunosuppressant rapamycin
Schwecke, T.; Aparicio, J. F.; Molnár, I. et al

in Proceedings of the National Academy of Sciences of the United States of America (1995), 92(17), 7839-7843

The macrocyclic polyketides rapamycin and FK506 are potent immunosuppressants that prevent T-cell proliferation through specific binding to intracellular protein receptors (immunophilins). The cloning and ... [more ▼]

The macrocyclic polyketides rapamycin and FK506 are potent immunosuppressants that prevent T-cell proliferation through specific binding to intracellular protein receptors (immunophilins). The cloning and specific alteration of the biosynthetic genes for these polyketides might allow the biosynthesis of clinically valuable analogues. We report here that three clustered polyketide synthase genes responsible for rapamycin biosynthesis in Streptomyces hygroscopicus together encode 14 homologous sets of enzyme activities (modules), each catalyzing a specific round of chain elongation. An adjacent gene encodes a pipecolate-incorporating enzyme, which completes the macrocycle. The total of 70 constituent active sites makes this the most complex multienzyme system identified so far. The DNA region sequenced (107.3 kbp) contains 24 additional open reading frames, some of which code for proteins governing other key steps in rapamycin biosynthesis. [less ▲]

Detailed reference viewed: 63 (3 UL)