Synthesis of peptide bonds by proteinases. Addition of organic cosolvents shifts peptide bond equilibriums toward synthesis

The extracellular siderophore from Mycobacterium smegmatis, exochelin MS, was isolated from iron-deficiently grown cultures and purified to > 98% by a combination of ion-exchange chromatography and h.p.l.c. The material is unextractable into organic solvents, is basic (pI = 9.3-9.5), has a lambda max at 420 nm and a probable Ks for Fe3+ of between 10(25) and 10(30). Its structure has been determined by examination of desferri- and ferri-exochelin and its gallium complex. The methods used were electrospray-m.s. and one- and two-dimensional (NOESY, DQF-COSY and TOCSY) 1H n.m.r. The constituent amino acids were examined by chiral g.l.c analysis of N-trifluoroacetyl isopropyl and N-pentafluoropropionyl methyl esters after hydrolysis, and reductive HI hydrolysis, of the siderophore. The exochelin is a formylated pentapeptide: N-(delta-N-formyl,delta N-hydroxy-R-ornithyl) -beta-alaninyl-delta N-hydroxy-R-ornithinyl-R-allo-threoninyl-delta N-hydroxy-S-ornithine. The linkages involving the three ornithine residues are via their delta N(OH) and alpha-CO groups leaving three free alpha-NH2 groups. Although there are two peptide bonds, these involve the three R (D)-amino acids. Thus the molecule has no conventional peptide bond, and this suggests that it will be resistant to peptidase hydrolysis. The co-ordination centre with Fe3+ is hexadenate in an octahedral structure involving the three hydroxamic acid groups. Molecular modelling shows it to have similar features to other ferric trihydroxamate siderophores whose three-dimensional structures have been established. The molecule is shown to have little flexibility around the iron chelation centre, although the terminal (Orn-3) residue, which is not involved in iron binding except at its delta N atom, has more motional freedom.

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The complete amino acid sequence of chicken skeletal-muscle enolase

Biochemical Journal ◽

10.1042/bj2360115 ◽

1986 ◽

Vol 236 (1) ◽

pp. 115-126 ◽

Cited By ~ 17

Author(s):

G A Russell ◽

B Dunbar ◽

L A Fothergill-Gilmore

Keyword(s):

Skeletal Muscle ◽

Amino Acid ◽

Amino Acid Sequence ◽

Peptide Bond ◽

Peptide Bonds ◽

Complete Amino Acid Sequence ◽

Arginine Residues ◽

Cnbr Cleavage ◽

Yeast Enolase ◽

Chicken Skeletal Muscle

The complete amino acid sequence of chicken skeletal-muscle enolase, comprising 433 residues, was determined. The sequence was deduced by automated sequencing of hydroxylamine-cleavage, CNBr-cleavage, o-iodosobenzoic acid-cleavage, clostripain-digest and staphylococcal-proteinase-digest fragments. The presence of several acid-labile peptide bonds and the tenacious aggregation of most CNBr-cleavage fragments meant that a commonly used sequencing strategy involving initial CNBr cleavage was unproductive. Cleavage at the single Asn-Gly peptide bond with hydroxylamine proved to be particularly useful. Comparison of the sequence of chicken enolase with the two yeast enolase isoenzyme sequences shows that the enzyme is strongly conserved, with 60% of the residues identical. The histidine and arginine residues implicated as being important for the activity of yeast enolase are conserved in the chicken enzyme. Secondary-structure predictions are analysed in an accompanying paper [Sawyer, Fothergill-Gilmore & Russell (1986) Biochem. J. 236, 127-130].

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Oxazolidinone Antibiotics Target the P Site on Escherichiacoli Ribosomes

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.4.1080-1085.2002 ◽

2002 ◽

Vol 46 (4) ◽

pp. 1080-1085 ◽

Cited By ~ 87

Author(s):

Hiroyuki Aoki ◽

Lizhu Ke ◽

Susan M. Poppe ◽

Toni J. Poel ◽

Elizabeth A. Weaver ◽

...

Keyword(s):

Antimicrobial Agents ◽

Wide Spectrum ◽

Anaerobic Bacteria ◽

Elongation Factor ◽

Peptide Bond ◽

23S Rrna ◽

Peptide Bonds ◽

A Site ◽

Domain V ◽

Trna Binding

ABSTRACT The oxazolidinones are a novel class of antimicrobial agents that target protein synthesis in a wide spectrum of gram-positive and anaerobic bacteria. The oxazolidinone PNU-100766 (linezolid) inhibits the binding of fMet-tRNA to 70S ribosomes. Mutations to oxazolidinone resistance in Halobacterium halobium, Staphylococcus aureus, and Escherichia coli map at or near domain V of the 23S rRNA, suggesting that the oxazolidinones may target the peptidyl transferase region responsible for binding fMet-tRNA. This study demonstrates that the potency of oxazolidinones corresponds to increased inhibition of fMet-tRNA binding. The inhibition of fMet-tRNA binding is competitive with respect to the fMet-tRNA concentration, suggesting that the P site is affected. The fMet-tRNA reacts with puromycin to form peptide bonds in the presence of elongation factor P (EF-P), which is needed for optimum specificity and efficiency of peptide bond synthesis. Oxazolidinone inhibition of the P site was evaluated by first binding fMet-tRNA to the A site, followed by translocation to the P site with EF-G. All three of the oxazolidinones used in this study inhibited translocation of fMet-tRNA. We propose that the oxazolidinones target the ribosomal P site and pleiotropically affect fMet-tRNA binding, EF-P stimulated synthesis of peptide bonds, and, most markedly, EF-G-mediated translocation of fMet-tRNA into the P site.

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Detection oftrans–cisflips and peptide-plane flips in protein structures

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715008263 ◽

2015 ◽

Vol 71 (8) ◽

pp. 1604-1614 ◽

Cited By ~ 16

Author(s):

Wouter G. Touw ◽

Robbie P. Joosten ◽

Gert Vriend

Keyword(s):

Structure Function ◽

Web Service ◽

Protein Data Bank ◽

Protein Structures ◽

Data Bank ◽

Peptide Bond ◽

Peptide Bonds ◽

Unknown Peptide ◽

Peptide Plane ◽

Service Interface

A coordinate-based method is presented to detect peptide bonds that need correction either by a peptide-plane flip or by atrans–cisinversion of the peptide bond. When applied to the whole Protein Data Bank, the method predicts 4617trans–cisflips and many thousands of hitherto unknown peptide-plane flips. A few examples are highlighted for which a correction of the peptide-plane geometry leads to a correction of the understanding of the structure–function relation. All data, including 1088 manually validated cases, are freely available and the method is available from a web server, a web-service interface and throughWHAT_CHECK.

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Recognition of protein-linked glycans as a determinant of peptidase activity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1615141114 ◽

2017 ◽

Vol 114 (5) ◽

pp. E679-E688 ◽

Cited By ~ 27

Author(s):

Ilit Noach ◽

Elizabeth Ficko-Blean ◽

Benjamin Pluvinage ◽

Christopher Stuart ◽

Meredith L. Jenkins ◽

...

Keyword(s):

Posttranslational Modifications ◽

Peptide Bond ◽

Peptidase Activity ◽

Peptide Bonds ◽

Crystallographic Structures ◽

Covalently Linked ◽

Biochemical Analyses ◽

Life On Earth ◽

Glycoprotein Degradation ◽

Hydrolysis Of

The vast majority of proteins are posttranslationally altered, with the addition of covalently linked sugars (glycosylation) being one of the most abundant modifications. However, despite the hydrolysis of protein peptide bonds by peptidases being a process essential to all life on Earth, the fundamental details of how peptidases accommodate posttranslational modifications, including glycosylation, has not been addressed. Through biochemical analyses and X-ray crystallographic structures we show that to hydrolyze their substrates, three structurally related metallopeptidases require the specific recognition of O-linked glycan modifications via carbohydrate-specific subsites immediately adjacent to their peptidase catalytic machinery. The three peptidases showed selectivity for different glycans, revealing protein-specific adaptations to particular glycan modifications, yet always cleaved the peptide bond immediately preceding the glycosylated residue. This insight builds upon the paradigm of how peptidases recognize substrates and provides a molecular understanding of glycoprotein degradation.

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Large facilities and the evolving ribosome, the cellular machine for genetic-code translation

Journal of The Royal Society Interface ◽

10.1098/rsif.2009.0167.focus ◽

2009 ◽

Vol 6 (suppl_5) ◽

Cited By ~ 20

Author(s):

Ada Yonath

Keyword(s):

Synchrotron Radiation ◽

Genetic Code ◽

Structural Information ◽

Peptide Bond ◽

Polymerase Activity ◽

Resistance Mechanisms ◽

Peptide Bond Formation ◽

Peptide Bonds ◽

Polypeptide Chains

Well-focused X-ray beams, generated by advanced synchrotron radiation facilities, yielded high-resolution diffraction data from crystals of ribosomes, the cellular nano-machines that translate the genetic code into proteins. These structures revealed the decoding mechanism, localized the mRNA path and the positions of the tRNA molecules in the ribosome and illuminated the interactions of the ribosome with initiation, release and recycling factors. They also showed that the ribosome is a ribozyme whose active site is situated within a universal symmetrical region that is embedded in the otherwise asymmetric ribosome structure. As this highly conserved region provides the machinery required for peptide bond formation and for ribosome polymerase activity, it may be the remnant of the proto-ribosome, a dimeric pre-biotic machine that formed peptide bonds and non-coded polypeptide chains. Synchrotron radiation also enabled the determination of structures of complexes of ribosomes with antibiotics targeting them, which revealed the principles allowing for their clinical use, revealed resistance mechanisms and showed the bases for discriminating pathogens from hosts, hence providing valuable structural information for antibiotics improvement.

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Characterization of meprin, a membrane-bound metalloendopeptidase from mouse kidney

Biochemical Journal ◽

10.1042/bj2410229 ◽

1987 ◽

Vol 241 (1) ◽

pp. 229-235 ◽

Cited By ~ 58

Author(s):

P E Butler ◽

M J McKay ◽

J S Bond

Keyword(s):

Amino Acid ◽

Degradation Products ◽

Amino Acid Content ◽

Peptide Bond ◽

Mouse Kidney ◽

Amino Acid Residues ◽

Peptide Bonds ◽

Membrane Bound ◽

Terminal Amino

Meprin is an intrinsic protein of the brush border, a specialized plasma membrane, of the mouse kidney. It is a metalloendopeptidase that contains 1 mol of zinc and 3 mol of calcium per mol of the 85,000-Mr subunit. The enzyme is isolated, and active, as a tetramer. The behaviour of the enzyme on SDS/polyacrylamide gels in the presence and absence of beta-mercaptoethanol indicates that the subunits are of the same Mr (approx. 85,000) and held together by intersubunit S–S bridges. Eight S-carboxymethyl-L-cysteine residues were detected after reduction of the enzyme with beta-mercaptoethanol and carboxymethylation with iodoacetate. The enzyme is a glycoprotein and contains approx. 18% carbohydrate. Most of the carbohydrate is removed by endoglycosidase F, indicating that the sugar residues are N-linked. The isoelectric point of the enzyme is between pH 4 and 5, and the purified protein yields a pattern of evenly spaced bands in this range on isoelectric focusing. The peptide-bond specificity of the enzyme has been determined by using the oxidized B-chain of insulin as substrate. In all, 15 peptide degradation products were separated by h.p.l.c. and analysed for their amino acid content and N-terminal amino acid residue. The prevalent peptide-bond cleavages were between Gly20 and Glu21, Phe24 and Phe25 and between Phe25 and Tyr26. Other sites of cleavage were Leu6-Cysteic acid7, Ala14-Leu15, His10-Leu11, Leu17-Val18, Gly8-Ser9, Leu15-Tyr16, His5-Leu6. These results indicate that meprin has a preference for peptide bonds that are flanked by hydrophobic or neutral amino acid residues, but hydrolysis is not limited to these bonds. The ability of meprin to hydrolyse peptide bonds between small neutral and negatively charged amino acid residues distinguishes it from several other metalloendopeptidases.

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Scandium(iii) triflate-promoted serine/threonine-selective peptide bond cleavage

Chemical Communications ◽

10.1039/c6cc10300f ◽

2017 ◽

Vol 53 (23) ◽

pp. 3311-3314 ◽

Cited By ~ 9

Author(s):

Jizhi Ni ◽

Youhei Sohma ◽

Motomu Kanai

Keyword(s):

Bond Cleavage ◽

Peptide Bond ◽

High Conversion ◽

Selective Hydrolysis ◽

Conversion Yield ◽

Peptide Bonds ◽

Peptide Bond Cleavage ◽

High Conversion Yield ◽

Site Selective ◽

Hydrolysis Of

The site-selective hydrolysis of peptide bonds at Ser and Thr positions was promoted by scandium(iii) triflate with a high conversion yield.

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On Ribosome Conservation and Evolution

Israel Journal of Ecology and Evolution ◽

10.1560/ijee_52_3-4_359 ◽

2006 ◽

Vol 52 (3-4) ◽

pp. 359-374 ◽

Cited By ~ 39

Author(s):

Ilana Agmon ◽

Anat Bashan ◽

Ada Yonath

Keyword(s):

Bond Formation ◽

Peptide Bond ◽

Binding Modes ◽

Peptide Bond Formation ◽

Peptidyl Transferase ◽

Peptide Bonds ◽

Peptidyl Transferase Center ◽

Enzymatic Function ◽

Rotatory Motion ◽

Rna Fold

The ribosome is a ribozyme whose active site, the peptidyl transferase center (PTC), is situated within a highly conserved universal symmetrical region that connects all ribosomal functional centers involved in amino acid polymerization. The linkage between this elaborate architecture and A-site tRNA position revealed that the A-> P-site passage of the tRNA terminus in the peptidyl transferase center is performed by a rotatory motion, synchronized with the overall tRNA/mRNA sideways movement. Guided by the PTC, the rotatory motion leads to stereochemistry suitable for peptide bond formation, as well as for substrate-mediated catalysis, consistent with quantum mechanical calculations elucidating the transition state mechanism for peptide bond formation and indicating that the peptide bond is being formed during the rotatory motion. Analysis of substrate binding modes to inactive and active ribosomes illuminated the significant PTC mobility and supported the hypothesis that the ancient ribosome produced single peptide bonds and non-coded chains, utilizing free amino acids. Genetic control of the reaction evolved after poly-peptides capable of enzymatic function were created, and an ancient stable RNA fold was converted into tRNA molecules. As the symmetry relates only the backbone fold and nucleotide orientations, but not nucleotide sequence, it emphasizes the superiority of functional requirement over sequence conservation, and indicates that the PTC has evolved by gene fusion, presumably by taking advantage of similar RNA fold structures.

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Effect of Mutations in the Human Immunoglobulin A1 (IgA1) Hinge on Its Susceptibility to Cleavage by Diverse Bacterial IgA1 Proteases

Infection and Immunity ◽

10.1128/iai.73.3.1515-1522.2005 ◽

2005 ◽

Vol 73 (3) ◽

pp. 1515-1522 ◽

Cited By ~ 29

Author(s):

Bernard W. Senior ◽

Jenny M. Woof

Keyword(s):

Peptide Bond ◽

Human Immunoglobulin ◽

Peptide Sequence ◽

Wild Type ◽

Peptide Bonds ◽

Streptococcus Sanguis ◽

In The Wild ◽

Scissile Bond ◽

Cleavable Peptide

ABSTRACT Components of the human immunoglobulin A1 (IgA1) hinge governing sensitivity to cleavage by bacterial IgA1 proteases were investigated. Recombinant antibodies with distinct hinge mutations were constructed from a hybrid comprised of human IgA2 bearing half of the human IgA1 hinge region. This hybrid antibody and all the mutant antibodies derived from it were resistant to cleavage by the IgA1 proteases from Streptococcus oralis and Streptococcus mitis biovar 1 strains but were cleaved to various degrees by those of Streptococcus pneumoniae, some Streptococcus sanguis strains, and the type 1 and 2 IgA1 proteases of Haemophilus influenzae, Neisseria meningitidis, and Neisseria gonorrhoeae. Remarkably, those proteases that cleave a Pro-Ser peptide bond in the wild-type IgA1 hinge were able to cleave mutant antibodies lacking a Pro-Ser peptide bond in the hinge, and those that cleave a Pro-Thr peptide bond in the wild-type IgA1 hinge were able to cleave mutant antibodies devoid of a Pro-Thr peptide bond in the hinge. Thus, the enzymes can cleave alternatives to their preferred postproline peptide bond when such a bond is unavailable. Peptide sequence analysis of a representative antibody digestion product confirmed this conclusion. The presence of a cleavable peptide bond near the CH2 end of the hinge appeared to result in greater cleavage than if the scissile bond was at the CH1 end of the hinge. Proline-to-serine substitution at residue 230 in a hinge containing potentially cleavable Pro-Ser and Pro-Thr peptide bonds increased the resistance of the antibody to cleavage by many IgA1 proteases.

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