Biomedical subjects

R Dieckmann

Publications and source records attributed to R Dieckmann.

8 recordsLinked to original sources

PubMed1999

Editing of non-cognate aminoacyl adenylates by peptide synthetases.

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 l-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-aromatic amino acids l-Leu and l-Met, or l-Phe analogues such as p-amino- and p-chloro-l-Phe derivatives, as well as the stereospecific selection of l-Phe, is supported by less-stable adenylate intermediates exhibiting elevated susceptibility to hydrolysis. Breakdown of the l-phenylalanyl intermediate utilizing 2'-deoxy-ATP as the nucleotide substrate was significantly enhanced compared with the natural analogue. Apo-TY1 engineered at positions involved in adenylate formation showed variable protection against hydrolysis. The results imply that stability of the aminoacyl intermediates may act as an essential factor in substrate selection and fidelity of non-ribosomal-peptide-forming systems.

Adenosine Monophosphate↗

PubMed1999

Probing the domain structure and ligand-induced conformational changes by limited proteolysis of tyrocidine synthetase 1.

The boundaries of the structural domains in peptide synthetases and the conformational changes related to catalysis were investigated by limited proteolysis of tyrocidine synthetase 1 (TY1). Four regions sensitive to proteolysis were detected (cleavage site at Arg13, Arg424, Arg509 and Arg602) that, in addition to an N-terminal extension, accurately delineate the domain boundaries of the adenylate-forming domain, the aminoacyl carrier domain, and the epimerisation domain. Limited proteolysis of an active N-terminal truncated deletion mutant, His6DeltaTY1, generated two stable and structurally independent subunits, corresponding to the subdomains of the adenylation domain. The structural integrity of the carrier domain was substantiated by its resistance to proteolytic degradation. Evidence is provided that the C-terminal "spacer" region with epimerising and/or condensing activity folds into an autonomous domain stable against degradation by limited proteoly sis. In the presence of substrates, reduced susceptibility to proteolysis was observed in the linker region connecting the subdomains of the adenylation domain, and corresponding to a peptide stretch of low electron density in the X-ray structure of the homologous firefly luciferase. Sequence analysis has shown that the respective linker contains conserved residues, whereas the linker regions connecting the structural domains are of low homology with a significant content of Pro, Ala, Glu and polar residues. A combination of kinetic and proteolytic studies using ATP analogues with substitutions in the phosphate chain, AMP-PcP, AMP-PNP and AMP-cPP, strongly suggests that the generation of a productive complex is associated with the ability of the beta, gamma-pyrophosphate moiety of ATP to adopt the proper active-site conformation. These data substantiate the observation that peptide synthetases undergo a series of conformational changes in the process of adenylate formation and product release.

Adenosine Triphosphate↗

PubMed1999

The nonribosomal code.

How genes are expressed and translated into proteins (using mRNA, codons and tRNAs as adaptor molecules) forms the basis of the 'genetic code'. Many peptides are synthesized nonribosomally, however, by large protein complexes that also serve as templates. Recent advances have shed light on what the nonribosomal code is and how it can be read.

Acylation↗

PubMed1998

Group specific antibodies against the putative AMP-binding domain signature SGTTGXPKG in peptide synthetases and related enzymes.

The superfamily of adenylate forming enzymes including peptide synthetases, acyl-CoA synthetases and insect luciferases is readily identified by the signature sequence SGTTGXPKG. This sequence including an invariant lysyl residue is located in a disordered loop region and was predicted to be of significant antigenicity. Antibodies were generated employing YTSGTTGRPKGC attached to bovine serum albumin and have been successfully used to identify respective enzymes and adenylate forming domains in multienzyme systems. These include the delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetases of Aspergillus nidulans and Acremonium chrysogenum, gramicidin S synthetase 1 and tyrocidine synthetase 1 from Bacillus brevis, acetyl-CoA synthetase from Alcaligenes eutrophus and a putative peptide synthetase from Metarhizium anisopliae. Weaker or no reactions are observed when the amino acid in position X in the protein is non-basic or hydrophobic, which is respectively the case for gramicidin S synthetase 1 and luciferase.

Adenosine Monophosphate↗

PubMed1997

ACV synthetase: expression of amino acid activating domains of the Penicillium chrysogenum enzyme in Aspergillus nidulans.

Fragments of ACV synthetase of Penicillium chrysogenum carrying partial activities of amino acid activation were expressed under the alcA promoter in an acvA-deletion mutant of Aspergillus nidulans. The 210 kDa domain A-beta-galactosidase fusion protein was partially cleaved to fragments of 200 and 97 kDa. The domain A fragment and the 312 kDa domain BC construct were identified by peptide specific antibodies and shown to catalyze alpha-aminoadipate-, cysteine-, and valine-dependent ATP/[32P]PPi exchange activity. Substrate specificities were investigated using amino acid analogues. Unexpectedly neither alpha-aminoadipate nor valine activation was exclusive, implying possible misactivations and proof reading functions. Both fragments were only expressed in limited amounts and found to be unstable.

Adenosine Triphosphate↗

PubMed1997

The adenylation domain of tyrocidine synthetase 1--structural and functional role of the interdomain linker region and the (S/T)GT(T/S)GXPKG core sequence.

Sequence analysis of peptide synthetases revealed extensive structure similarity with firefly luciferase, whose crystal structure has recently become available, providing evidence for the localization of the active site at the interface between two subdomains separated by a distorted linker region [Conti, E., Franks, N. P. & Brick, P. (1996) Structure 4, 287-298]. The functional importance of two flexible loops, corresponding to the linker region of firefly luciferase and the highly conserved (S/T)GT(T/S)GXPKG core sequence, has been studied in view of the proposed conformational changes by the use of mutant analysis, limited proteolysis and chemical modification of tyrocidine synthetase 1. Substitution of the highly conserved Arg416, residing in the loop separating the subdomains of the adenylation domain, resulted in profound loss of activity. Limited proteolysis of the mutant suggested significant structural changes as manifested by lack of protection to degradation in the presence of substrates, revealing a probable disturbance of the induced-fit mechanism regulating the transformation from an open to a closed conformation. Mutants, obtained by replacement of the conserved Lys186 from the (S/T)GT(T/S)GXPKG core sequence, displayed only minor differences in substrate-binding affinity despite significant reduction of catalytic efficiency. Residue Lys186 appears to play an important role in either stabilization of the bound substrate through charge-charge-interactions, and/or fixing of the loop for maintainance of the active-site conformation.

Amino Acid Sequence↗

PubMed1995

Expression of an active adenylate-forming domain of peptide synthetases corresponding to acyl-CoA-synthetases.

Peptide synthetases and acyl-CoA-synthetases form acyl adenylates which are transferred to CoA or enzyme-bound pantetheine. To verify the existence of an adenylate domain in peptide synthetases, a 60.8 kDa fragment of tyrocidine 1-synthetase was constructed by a 1,629 bp deletion, expressed in Escherichia coli, and characterized. The truncated multienzyme activated phenylalanine and substrate analogues with comparable kinetics as the over-expressed synthetase, as judged by ATP-[32P]PP(i) exchange reaction. Thus the N-terminal domain resembling an acyl-CoA-synthetase is an autonomous structural element. This N-terminal domain is followed by a cofactor binding domain, resembling acyl carrier proteins involved in polyketide formation.

Adenosine Monophosphate↗

PubMed1992

Safety of pre-hospital therapy with morphine sulfate.

The safety of prehospital pharmacologic therapy has not been well studied. The authors evaluated field use of morphine sulfate (MS) in San Francisco County over a 6-month period. Paramedics assessed patients for ischemic chest pain (ICP) and/or pulmonary edema (PE), made base hospital contact, and administered 2- to 4-mg doses of intravenous morphine according to treatment protocols. Clinical assessments and patient responses to therapy were recorded by both field paramedics and emergency department (ED) physicians. Safety was evaluated by determining the (1) accuracy of paramedic field assessment, (2) appropriateness of field administration of MS, and (3) therapeutic complications. During the study period, paramedics administered MS to 84 patients. In 69 cases paramedic assessment of either ICP and/or PE corresponded to ED physician diagnosis. In five cases paramedics correctly recognized ICP but missed physical findings of PE. In this group the paramedics' assessment was considered inaccurate but the judgement to give MS was considered appropriate. In the remaining 10 cases paramedics identified ICP or PE but the ED physician diagnosed a different condition. These assessments were considered inaccurate and the management inappropriate. Therefore, overall paramedic accuracy was 77% (true rate 73% to 82%, 95% confidence interval); appropriateness of therapy was 88% (true rate 85% to 92%, 95% confidence interval); and the overall complication rate was 6% (true rate 2% to 12%, 95% confidence interval). Complications of respiratory depression or hypotension occurred in only one of the cases in which MS was inappropriately administered.(ABSTRACT TRUNCATED AT 250 WORDS)

Chest Pain↗