Title: Enzymatic reconstitution of ribosomal peptide backbone thioamidation

Abstract

Methyl-coenzyme M reductase (MCR) is an essential enzyme found strictly in methanogenic and methanotrophic archaea. MCR catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The α-subunit of this enzyme (McrA) contains several unusual posttranslational modifications, including the only known naturally occurring example of protein thioamidation. We have recently demonstrated by genetic deletion and mass spectrometry that the tfuA and ycaO genes of Methanosarcina acetivorans are involved in thioamidation of Gly465 in the MCR active site. Modification to thioGly has been postulated to stabilize the active site structure of MCR. Herein, we report the in vitro reconstitution of ribosomal peptide thioamidation using heterologously expressed and purified YcaO and TfuA proteins from M. acetivorans. Like other reported YcaO proteins, this reaction is ATP-dependent but requires an external sulfide source. We also reconstitute the thioamidation activity of two TfuA-independent YcaOs from the hyperthermophilic methanogenic archaea Methanopyrus kandleri and Methanocaldococcus jannaschii. Using these proteins, we demonstrate the basis for substrate recognition and regioselectivity of thioamide formation based on extensive mutagenesis, biochemical, and binding studies. Finally, we report nucleotide-free and nucleotide-bound crystal structures for the YcaO proteins from M. kandleri. Sequence and structure-guided mutagenesis with subsequent biochemicalmore » evaluation have allowed us to assign roles for residues involved in thioamidation and confirm that the reaction proceeds via backbone O-phosphorylation. We conclude these data assign a new biochemical reaction to the YcaO superfamily and paves the way for further characterization of additional peptide backbone posttranslational modifications.« less

Authors:

Mahanta, Nilkamal ^[1]; Liu, Andi ^[1]; Dong, Shihui  ^[1];

• Search DOE PAGES for author "Dong, Shihui"
• Search DOE PAGES for ORCID "0000-0002-1743-2163"

Nair, Satish K. ^[1]; Mitchell, Douglas A.  ^[1]

• Search DOE PAGES for author "Mitchell, Douglas A."
• Search DOE PAGES for ORCID "0000-0002-9564-0953"

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1. Univ. of Illinois at Urbana-Champaign, IL (United States)

Publication Date:

Mon Mar 05 00:00:00 EST 2018

Research Org.:

Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Org.:

National Institutes of Health (NIH)

OSTI Identifier:

1499738

Grant/Contract Number:  

GM097142

Resource Type:

Accepted Manuscript

Journal Name:

Proceedings of the National Academy of Sciences of the United States of America

Additional Journal Information:

Journal Volume: 115; Journal Issue: 12; Journal ID: ISSN 0027-8424

Publisher:

National Academy of Sciences

Country of Publication:

United States

Language:

ENGLISH

Subject:

59 BASIC BIOLOGICAL SCIENCES; thioamide; methanogen; posttranslational modification; YcaO; methyl-coenzyme M reductase