scholarly journals Evolutionary and functional analysis of an NRPS condensation domain integrates β-lactam, ᴅ-amino acid, and dehydroamino acid synthesis

2021 ◽  
Vol 118 (17) ◽  
pp. e2026017118
Author(s):  
Michael J. Wheadon ◽  
Craig A. Townsend
Keyword(s):  
Natural Products ◽  
Amino Acid ◽  
Bond Formation ◽  
Immediate Release ◽  
Peptide Bond ◽  
Sequence Motifs ◽  
Peptide Bond Formation ◽  
Specific Sequence ◽  
Dehydroamino Acid

Nonribosomal peptide synthetases (NRPSs) are large, multidomain biosynthetic enzymes involved in the assembly-line–like synthesis of numerous peptide natural products. Among these are clinically useful antibiotics including three classes of β-lactams: the penicillins/cephalosporins, the monobactams, and the monocyclic nocardicins, as well as the vancomycin family of glycopeptides and the depsipeptide daptomycin. During NRPS synthesis, peptide bond formation is catalyzed by condensation (C) domains, which couple the nascent peptide with the next programmed amino acid of the sequence. A growing number of additional functions are linked to the activity of C domains. In the biosynthesis of the nocardicins, a specialized C domain prepares the embedded β-lactam ring from a serine residue. Here, we examine the evolutionary descent of this unique β-lactam–synthesizing C domain. Guided by its ancestry, we predict and demonstrate in vitro that this C domain alternatively performs peptide bond formation when a single stereochemical change is introduced into its peptide starting material. Remarkably, the function of the downstream thioesterase (TE) domain also changes. Natively, the TE directs C terminus epimerization prior to hydrolysis when the β-lactam is made but catalyzes immediate release of the alternative peptide. In addition, we investigate the roles of C-domain histidine residues in light of clade-specific sequence motifs, refining earlier mechanistic proposals of both β-lactam formation and canonical peptide synthesis. Finally, expanded phylogenetic analysis reveals unifying connections between β-lactam synthesis and allied C domains associated with the appearance of ᴅ-amino acid and dehydroamino acid residues in other NRPS-derived natural products.

Oxidative Peptide Bond Formation of Glycine-Amino Acid using 2-(Aminomethyl)malononitrile as a Glycine Unit

2021 ◽  
Author(s):  
Xiaoling Wang ◽  
Jing Li ◽  
Yujiro Hayashi
Keyword(s):  
Aqueous Solution ◽  
Amino Acid ◽  
Bond Formation ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Amide Linkage

Amide linkage of glycine-amino acid was synthesized by coupling of substituted 2-(aminomethyl)malononitrile as a C-terminal glycine unit and N-terminal amine using CsOAc and O2 in aqueous solution. This is a...

scholarly journals Post-translational peptide bond formation during concanavalin A processing in vitro

1996 ◽  
Vol 320 (3) ◽  
pp. 865-870 ◽  
Author(s):  
Philip S. SHELDON ◽  
Jeffrey N. KEEN ◽  
Dianna J. BOWLES
Keyword(s):  
Concanavalin A ◽  
De Novo ◽  
Bond Formation ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Con A ◽  
Glycoprotein Precursor ◽  
Tyrosine Residues ◽  
Processing Activity

Post-translational processing of concanavalin A (Con A) is complex, involving deglycosylation, proteolytic cleavage on the carboxy group side of asparagine residues and formation of a peptide bond de novo. This has been studied with the 125I-labelled Con A glycoprotein precursor as a substrate for processing in vitro. Extracts of immature jackbean cotyledons and the commercially available purified preparation of asparaginylendopeptidase were able to catalyse the above processes. The processing resulted in the conversion of the 33.5 kDa inactive glycoprotein precursor into an active lectin. Processing activity was maximal at approx. pH 5.5. Evidence to support processing at authentic sites was obtained by observation of the release of 125I at positions in the sequence where tyrosine residues were present.

scholarly journals Abstract P-29: Cryoem Study of the Inhibition of Bacterial Ribosomes by Madumycin II

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S24-S25
Author(s):  
Alena Yakusheva ◽  
Olga Shulenina ◽  
Evgeny Pichkur ◽  
Alena Paleskava ◽  
Alexander Myasnikov ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Resolution ◽  
Bond Formation ◽  
Protein Translation ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
70S Ribosome ◽  
Madumycin Ii

Background: The efficiency of widely used antibiotics is limited by continuous improvement of resistance mechanisms. Thus, the research of poorly studied drugs that have not received practical use until now becomes relevant again. Protein translation is one of the major targets for antibiotics. Madumycin II (MADU) is an antibiotic of the streptogramin A class that binds to the peptidyl transferase center of the initiated bacterial 70S ribosome inhibiting the first cycle of peptide bond formation (I.A. Osterman et al. Nucleic Acids Res., 2017). The ability of MADU to interfere with translating ribosome is an open question that we address by investigation of high-resolution cryo-EM structures of MADU bound 70S ribosome complexes from Escherichia coli. Methods: Purified initiated and translating ribosome complexes preincubated with MADU were applied onto freshly glow discharged carbon-coated grids (Quantifoil R 1.2/1.3) and flash-frozen in the liquid ethane pre-cooled by liquid nitrogen in the Vitrobot Mark IV. Frozen grids were transferred into an in-house Titan Krios microscope. Data were collected using EPU software. Movie stacks were preprocessed in Warp software. For image processing, we have used several software packages: Relion 3.1, CryoSPARC, and CisTEM. The model was built in Coot. Results: We have obtained high-resolution cryo-EM structures of two ribosomal complexes with MADU before and after the first cycle of peptide bond formation with an average resolution of 2.3 Å. Preliminary analysis of the structures shows no major differences in the MADU binding mode to the ribosomal complexes under study suggesting that the quantity of amino acid residues attached to the P-site tRNA does not impact MADU bonding. Moreover, in both cases, we observed similar destabilization of the CCA-ends of A- and P-site tRNAs underlining the comparable influence of MADU on the ribosomal complexes. Conclusion: Our results suggest that although MADU binding site is located in the peptidyl transferase center, the presence of the second amino acid residue on the P-site tRNA does not preclude antibiotic binding. We assume that further elongation of the polypeptide chain would not have any impact either. High conformational lability of the CCA-ends of tRNA at the A and P sites upon binding of MADU obviously plays an important role in the inhibition mechanism of the bacterial ribosome. The further structural and biochemical analysis will be necessary to shed more light on the detailed mechanism of MADU action.

Post-microsomal fractions from rat brain which stimulate ribosomal amino acid incorporation

1969 ◽  
Vol 47 (2) ◽  
pp. 75-78 ◽  
Author(s):  
M. R. V. Murthy
Keyword(s):  
Amino Acid ◽  
Rat Brain ◽  
Bond Formation ◽  
Peptide Bond ◽  
Sucrose Density Gradient ◽  
Peptide Bond Formation ◽  
Amino Acid Incorporation ◽  
Transfer Rnas ◽  
Complete Inactivation ◽  
Acid Incorporation

Post-microsomal fractions from rat brain were prepared which stimulated the synthesis of protein when endogenous mRNA was available and enhanced the synthesis of polyphenylalanine in the presence of poly-U and monomeric ribosomes. The amino-acid-activating (or transfer) enzymes did not appear to be responsible for the observed effect since the reaction mixture contained sufficient amounts of these enzymes to permit an optimum rate of amino acid incorporation. The effect was not solely due to the presence of messenger or amino acid transfer RNAs since the activity of the fractions was only partly reduced by prior treatment with ribonuclease. Heating led to complete inactivation of the fractions. Results from amino-acid-incorporation experiments and from sucrose density-gradient centrifugations indicate that the effect might be produced at two levels, one at the level of formation or preservation of polyribosomal structure and the other possibly at the level of peptide bond formation.

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