DOCSLIB.ORG

Site Directed Mutagenesis of L-Pyrrolysine in Dimethylamine Methyltransferase

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Site Directed Mutagenesis of L-Pyrrolysine in Dimethylamine Methyltransferase Site directed mutagenesis of L-pyrrolysine in dimethylamine methyltransferase A Senior Honors Thesis Presented in Partial Fulfillment of the Requirements for graduation with research distinction in Microbiology in the undergraduate colleges of The Ohio State University by Jigar Patel The Ohio State University March 2009 Project Advisor: Professor Joseph Krzycki, Department of Microbiology 1 Abstract Methanosarcina barkeri is an archaeon and is able to use methylamines as growth substrates for methanogenesis. In M. barkeri, the three methylamine methyltransferases that are involved in initiating methanogenesis from the methylamines have an in-frame amber codon (UAG) that does not terminate translation, but in fact encodes for the 22nd amino acid L- pyrrolysine. This thesis attempts to examine the rationale for the presence of pyrrolysine in one of these three methylamine methyltransferases. A system that allowed the recombinant expression of the dimethylamine methyltransferase gene, mtbB, in the native organism Methanosarcina acetivorans, was developed (9). Site directed mutagenesis of mtbB was performed wherein UAG, which encodes for pyrrolysine, was substituted by GCA, which encodes for alanine (Jodie Lee). This allowed for functional studies of MtbB vs. the mutant MtbB, dubbed MtbB(O356A). An activity assay, which measured the ability of MtbB and MtbB(O356A) to methylate hydroxocobalamin, showed that MtbB(O356A) had lost its functionality. This indicated that pyrrolysine is an essential residue in MtbB and that it may play a catalytic role. However, preliminary data from a native gel electrophoresis experiment showed that the ability of MtbB(O356A) to bind its cognate corrinoid cofactor protein, MtbC, was greatly reduced; whilst the wild-type MtbB was able to form a complex with MtbC. This indicated that pyrrolysine is required for proper folding of MtbB and/or it is required for binding MtbC. 2 Table of Contents Abstract ......................................................................................................................................................... 2 1. Introduction ............................................................................................................................................... 4 1.1 Methanogens ...................................................................................................................................... 4 1.2 Methanosarcina barkeri MS and L-pyrrolysine ................................................................................... 4 1.3 Pyrrolysine and methanogenesis in M.barkeri ................................................................................... 5 1.4 Proposed role of pyrrolysine in methanogenesis ............................................................................... 5 1.5 Project aim and hypothesis ................................................................................................................. 6 2. Materials and Methods ............................................................................................................................ 10 2.1 General Analytical Methods.............................................................................................................. 10 2.1.1 Denaturing polyacrylamide gel electrophoresis ......................................................................... 10 2.1.2 Protein assay .............................................................................................................................. 10 2.2 Isolation of MtbB .............................................................................................................................. 10 2.3 Isolation of MtbB(O356A) ................................................................................................................. 11 2.4 Isolation of MtbC .............................................................................................................................. 11 3. Assays ...................................................................................................................................................... 13 3.1 DMA-dependent cobalamin methylation by MtbB or MtbB(O356A) ............................................... 13 3.2 Methylation of MtbC by MtbB or MtbB(O356A) .............................................................................. 13 3.3 Visualization of MtbBC complex formation by native gel electrophoresis ....................................... 14 4. Results ..................................................................................................................................................... 15 4.1 Protein purification ........................................................................................................................... 15 4.2 MtbB (O356A) and wild type MtbB differ markedly in methylating cob(I)alamin ........................... 25 4.3 Assay of direct methylation of MtbC is incomplete because of inability to maintain the corrinoid cofactor of MtbC in the Co(I) state ......................................................................................................... 28 4.4 MtbB, but not MtbB(O356A) is able to form a putative complex with conjugate corrinoid protein MtbC ....................................................................................................................................................... 28 5. Discussion ............................................................................................................................................... 31 Bibliography ............................................................................................................................................... 34 3 1. Introduction 1.1 Methanogens Methanogens belong to the domain Archaea and help breakdown bacterial fermentation products during environmental recycling of organic nutrients. They do so by the process of methanogenesis, by which methanogens produce about 1 billion tons of methane per year. As a green house gas, this methane has an impact on our climate. Methanogens are also found in variety of environments, such as sanitary landfills, fresh and saline sediments, and animal gastro- intestinal tracts. Hence, methanogens play an important role in our ecosystem. 1.2 Methanosarcina barkeri MS and L-pyrrolysine M. barkeri is one such methanogen. But unlike many other methanogens, which thrive with canonical twenty amino acids, M. barkeri employs a rare amino acid in its enzymes that are involved in methanogenesis from methylamines. This amino acid is pyrrolysine. When the genetic code was solved, it was determined that the sequence of nucleotides in the mRNA molecule is read sequentially in groups of three nucleotides (a codon). Subsequently it was shown that the 64 possible nucleotide combinations either encode one of the known 20 common amino acids or terminate translation. It was also realized that the genetic code is universal. At this time, it was believed that the ochre, amber and the opal codons universally lead to translation termination. However, this perception has changed. It is now known that some organisms, under certain circumstances, can in fact code for amino acids using the stop codons. Amino acid selenocysteine, encoded by the opal codon UGA, was the first such example. 4 Interestingly, it was discovered that the genes encoding methylamine methyltransferases in M. barkeri employ an in-frame amber codon (UAG) as a sense codon as well. Subsequent evidence for a dedicated pyrrolysyl-t-RNA synthetase (PylS) and tRNAPyl, and the unique electron density of the amber-codon encoded amino acid led to and proved the existence of the 22nd amino acid, L-pyrrolysine (1; 2; 3; 4). Figure 1 shows the structure of L-pyrrolysine. It is a modified lysine with an amide linkage to (4R,5R)-4-methyl-pyrroline-5-carboxylate. 1.3 Pyrrolysine and methanogenesis in M.barkeri Pyrrolysine is found in the three methylamine methyltransferases in M. barkeri. Monomethylamine methyltransferase (MtmB), dimethylamine methyltransferase (MtbB) and trimethylamine methyltransferase (Mttb) each has a single residue of pyrrolysine incorporated into their sequence. As shown in Figure 2, MtmB, MtbB, and MttB are specific for their substrates monomethylamine (MMA), dimethylamine (DMA) and trimethylamine (TMA) respectively. Once substrate is bound, the methylated MtmB, MtbB or MttB methylate their cognate corrinoid proteins MtmC, MtbC or MttC respectively. Methylated MtmC, MtbC and MttC serve as substrates for MtbA, a methylcobamide:coenzyme M methyltransferase. MtbA demethylates the corrinoid proteins using zinc to form a nucleophilic coenzyme M thiolate, which, when reduced by methyl-CoM reductase, forms methane (5; 6; 7). 1.4 Proposed role of pyrrolysine in methanogenesis A crystal structure of MtmB solved to 1.7 Å suggests that pyrrolysine may play a catalytic role. The structure depicts pyrrolysine at the bottom of a negatively charged cleft. Additionally, pyrrolysine reacts with nucleophilic compounds such as hydroxylamine (17). 5 Based on these observations Hao et al proposed a possible mechanism for the role of pyrrolysine in methylation of MtmC (2). The mechanism is described in Figure 3. 1.5 Project aim and hypothesis In order to test the role of pyrrolysine in catalysis, the activity of wild-type MtbB was compared to that of a site-directed mutant MtbB, dubbed MtbB(O356A); wherein pyrrolysine encoded by codon 356 (UAG) was substituted with alanine (encoded by codon GCA).
Load more

Recommended publications
  • Selenocysteine, Pyrrolysine, and the Unique Energy Metabolism of Methanogenic Archaea
    Hindawi Publishing Corporation Archaea Volume 2010, Article ID 453642, 14 pages doi:10.1155/2010/453642 Review Article Selenocysteine, Pyrrolysine, and the Unique Energy Metabolism of Methanogenic Archaea Michael Rother1 and Joseph A. Krzycki2 1 Institut fur¨ Molekulare Biowissenschaften, Molekulare Mikrobiologie & Bioenergetik, Johann Wolfgang Goethe-Universitat,¨ Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany 2 Department of Microbiology, The Ohio State University, 376 Biological Sciences Building 484 West 12th Avenue Columbus, OH 43210-1292, USA Correspondence should be addressed to Michael Rother, [email protected] andJosephA.Krzycki,[email protected] Received 15 June 2010; Accepted 13 July 2010 Academic Editor: Jerry Eichler Copyright © 2010 M. Rother and J. A. Krzycki. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Methanogenic archaea are a group of strictly anaerobic microorganisms characterized by their strict dependence on the process of methanogenesis for energy conservation. Among the archaea, they are also the only known group synthesizing proteins containing selenocysteine or pyrrolysine. All but one of the known archaeal pyrrolysine-containing and all but two of the confirmed archaeal selenocysteine-containing protein are involved in methanogenesis. Synthesis of these proteins proceeds through suppression of translational stop codons but otherwise the two systems are fundamentally different. This paper highlights these differences and summarizes the recent developments in selenocysteine- and pyrrolysine-related research on archaea and aims to put this knowledge into the context of their unique energy metabolism. 1. Introduction found to correspond to pyrrolysine in the crystal structure [9, 10] and have its own tRNA [11].
    [Show full text]
  • Amino Acid Recognition by Aminoacyl-Trna Synthetases
    www.nature.com/scientificreports OPEN The structural basis of the genetic code: amino acid recognition by aminoacyl‑tRNA synthetases Florian Kaiser1,2,4*, Sarah Krautwurst3,4, Sebastian Salentin1, V. Joachim Haupt1,2, Christoph Leberecht3, Sebastian Bittrich3, Dirk Labudde3 & Michael Schroeder1 Storage and directed transfer of information is the key requirement for the development of life. Yet any information stored on our genes is useless without its correct interpretation. The genetic code defnes the rule set to decode this information. Aminoacyl-tRNA synthetases are at the heart of this process. We extensively characterize how these enzymes distinguish all natural amino acids based on the computational analysis of crystallographic structure data. The results of this meta-analysis show that the correct read-out of genetic information is a delicate interplay between the composition of the binding site, non-covalent interactions, error correction mechanisms, and steric efects. One of the most profound open questions in biology is how the genetic code was established. While proteins are encoded by nucleic acid blueprints, decoding this information in turn requires proteins. Te emergence of this self-referencing system poses a chicken-or-egg dilemma and its origin is still heavily debated 1,2. Aminoacyl-tRNA synthetases (aaRSs) implement the correct assignment of amino acids to their codons and are thus inherently connected to the emergence of genetic coding. Tese enzymes link tRNA molecules with their amino acid cargo and are consequently vital for protein biosynthesis. Beside the correct recognition of tRNA features3, highly specifc non-covalent interactions in the binding sites of aaRSs are required to correctly detect the designated amino acid4–7 and to prevent errors in biosynthesis5,8.
    [Show full text]
  • Direct Charging of Trnacua with Pyrrolysine in Vitro and in Vivo
    letters to nature .............................................................. gene product (see Supplementary Fig. S1). The tRNA pool extracted from Methanosarcina acetivorans or tRNACUA transcribed in vitro Direct charging of tRNACUA with was used in charging experiments. Charged and uncharged tRNA species were separated by electrophoresis in a denaturing acid-urea pyrrolysine in vitro and in vivo 10,11 polyacrylamide gel and tRNACUA was specifically detected by northern blotting with an oligonucleotide probe. The oligonucleo- Sherry K. Blight1*, Ross C. Larue1*, Anirban Mahapatra1*, tide complementary to tRNA could hybridize to a tRNA in the David G. Longstaff1, Edward Chang1, Gang Zhao2†, Patrick T. Kang4, CUA Kari B. Green-Church5, Michael K. Chan2,3,4 & Joseph A. Krzycki1,4 pool of tRNAs isolated from wild-type M. acetivorans but not to the tRNA pool from a pylT deletion mutant of M. acetivorans (A.M., 1Department of Microbiology, 484 West 12th Avenue, 2Department of Chemistry, A. Patel, J. Soares, R.L. and J.A.K., unpublished observations). 3 100 West 18th Avenue, Department of Biochemistry, 484 West 12th Avenue, Both tRNACUA and aminoacyl-tRNACUA were detectable in the The Ohio State University, Columbus, Ohio 43210, USA isolated cellular tRNA pool (Fig. 1). Alkaline hydrolysis deacylated 4Ohio State University Biochemistry Program, 484 West 12th Avenue, The Ohio the cellular charged species, but subsequent incubation with pyrro- State University, Columbus, Ohio 43210, USA lysine, ATP and PylS-His6 resulted in maximal conversion of 50% of 5CCIC/Mass Spectrometry and Proteomics Facility, The Ohio State University, deacylated tRNACUA to a species that migrated with the same 116 W 19th Ave, Columbus, Ohio 43210, USA electrophoretic mobility as the aminoacyl-tRNACUA present in the * These authors contributed equally to this work.
    [Show full text]
  • Site-Specific Protein Modifications Through Pyrroline-Carboxy-Lysine Residues
    Site-specific protein modifications through pyrroline-carboxy-lysine residues Weijia Ou1, Tetsuo Uno1, Hsien-Po Chiu, Jan Grünewald, Susan E. Cellitti, Tiffany Crossgrove, Xueshi Hao, Qian Fan, Lisa L. Quinn, Paula Patterson, Linda Okach, David H. Jones, Scott A. Lesley, Ansgar Brock, and Bernhard H. Geierstanger2 Genomics Institute of the Novartis Research Foundation, 10675 John-Jay-Hopkins Drive, San Diego, CA 92121-1125 Edited* by Peter G. Schultz, The Scripps Research Institute, La Jolla, CA, and approved May 11, 2011 (received for review April 4, 2011) Pyrroline-carboxy-lysine (Pcl) is a demethylated form of pyrrolysine acids will face similar hurdles to achieve site-specificity without that is generated by the pyrrolysine biosynthetic enzymes when deleterious effects to the protein of interest. the growth media is supplemented with D-ornithine. Pcl is readily The most elegant way to generate homogenously, site-specifi- incorporated by the unmodified pyrrolysyl-tRNA/tRNA synthetase cally modified proteins is the in vivo incorporation of unnatural pair into proteins expressed in Escherichia coli and in mammalian amino acids (7–9). Over 70 unnatural amino acids featuring a cells. Here, we describe a broadly applicable conjugation chemistry wide array of functionalities can be incorporated at TAG codons that is specific for Pcl and orthogonal to all other reactive groups using specific tRNA/tRNA synthetase pairs engineered through on proteins. The reaction of Pcl with 2-amino-benzaldehyde or an in vivo selection process to be orthogonal to the cellular 2-amino-acetophenone reagents proceeds to near completion at machinery of the host cells. A set of reactive unnatural amino neutral pH with high efficiency.
    [Show full text]
  • A 22Nd Amino Acid Encoded Through a Genetic Code Expansion
    Emerging Topics in Life Sciences (2018) https://doi.org/10.1042/ETLS20180094 Review Article Pyrrolysine in archaea: a 22nd amino acid encoded through a genetic code expansion Jean-François Brugère1,2, John F. Atkins3,4, Paul W. O’Toole2 and Guillaume Borrel5 1Université Clermont Auvergne, Clermont-Ferrand F-63000, France; 2School of Microbiology and APC Microbiome Institute, University College Cork, Cork, Ireland; 3School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland; 4Department of Human Genetics, University of Utah, Salt Lake City, UT, U.S.A.; 5Evolutionary Biology of the Microbial Cell, Department of Microbiology, Institut Pasteur, Paris, France Correspondence: Jean-François Brugère ( [email protected]) The 22nd amino acid discovered to be directly encoded, pyrrolysine, is specified by UAG. Until recently, pyrrolysine was only known to be present in archaea from a methanogenic lineage (Methanosarcinales), where it is important in enzymes catalysing anoxic methyla- mines metabolism, and a few anaerobic bacteria. Relatively new discoveries have revealed wider presence in archaea, deepened functional understanding, shown remark- able carbon source-dependent expression of expanded decoding and extended exploit- ation of the pyrrolysine machinery for synthetic code expansion. At the same time, other studies have shown the presence of pyrrolysine-containing archaea in the human gut and this has prompted health considerations. The article reviews our knowledge of this fascinating exception to the ‘standard’ genetic code. Introduction It is now more than half a century ago since the genetic code was deciphered [1,2]. The understanding gained of how just four different nucleobases could specify the 20 universal amino acids to synthesise effective proteins reveals a fundamental feature of extant life.
    [Show full text]
  • Proposal of the Annotation of Phosphorylated Amino Acids and Peptides Using Biological and Chemical Codes
    molecules Article Proposal of the Annotation of Phosphorylated Amino Acids and Peptides Using Biological and Chemical Codes Piotr Minkiewicz * , Małgorzata Darewicz , Anna Iwaniak and Marta Turło Department of Food Biochemistry, University of Warmia and Mazury in Olsztyn, Plac Cieszy´nski1, 10-726 Olsztyn-Kortowo, Poland; [email protected] (M.D.); [email protected] (A.I.); [email protected] (M.T.) * Correspondence: [email protected]; Tel.: +48-89-523-3715 Abstract: Phosphorylation represents one of the most important modifications of amino acids, peptides, and proteins. By modifying the latter, it is useful in improving the functional properties of foods. Although all these substances are broadly annotated in internet databases, there is no unified code for their annotation. The present publication aims to describe a simple code for the annotation of phosphopeptide sequences. The proposed code describes the location of phosphate residues in amino acid side chains (including new rules of atom numbering in amino acids) and the diversity of phosphate residues (e.g., di- and triphosphate residues and phosphate amidation). This article also includes translating the proposed biological code into SMILES, being the most commonly used chemical code. Finally, it discusses possible errors associated with applying the proposed code and in the resulting SMILES representations of phosphopeptides. The proposed code can be extended to describe other modifications in the future. Keywords: amino acids; peptides; phosphorylation; phosphate groups; databases; code; bioinformatics; cheminformatics; SMILES Citation: Minkiewicz, P.; Darewicz, M.; Iwaniak, A.; Turło, M. Proposal of the Annotation of Phosphorylated Amino Acids and Peptides Using 1. Introduction Biological and Chemical Codes.
    [Show full text]
  • Site-Specific Incorporation of Unnatural Amino Acids Into Escherichia Coli Recombinant Protein: Methodology Development and Recent Achievement
    Review Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement Sviatlana Smolskaya 1,* and Yaroslav A. Andreev 1,2 1 Sechenov First Moscow State Medical University, Institute of Molecular Medicine, Trubetskaya str. 8, bld. 2, 119991 Moscow, Russia 2 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho- Maklaya 16/10, 117997 Moscow, Russia; [email protected] * Correspondence: [email protected]; Tel.: +7-903-215-44-89 Received: 30 May 2019; Accepted: 25 June 2019; Published: 28 June 2019 Abstract: More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E.
    [Show full text]
  • A Natural Genetic Code Expansion Cassette Enables Transmissible Biosynthesis and Genetic Encoding of Pyrrolysine
    A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine David G. Longstaff*, Ross C. Larue*, Joseph E. Faust*, Anirban Mahapatra*, Liwen Zhang†, Kari B. Green-Church†, and Joseph A. Krzycki*‡§ *Department of Microbiology, †Campus Chemical Instrument Center/Mass Spectrometry and Proteomics Facility, and ‡Ohio State University Biochemistry Program, Ohio State University, 484 West 12th Avenue, Columbus, OH 43210 Communicated by David L. Denlinger, Ohio State University, Columbus, OH, November 20, 2006 (received for review September 14, 2006) Pyrrolysine has entered natural genetic codes by the translation of UAG, a canonical stop codon. UAG translation as pyrrolysine requires the pylT gene product, an amber-decoding tRNAPyl that is aminoacylated with pyrrolysine by the pyrrolysyl-tRNA synthetase produced from the pylS gene. The pylTS genes form a gene cluster with pylBCD, whose functions have not been investigated. The pylTSBCD gene order is maintained not only in methanogenic Fig. 1. The pyl genes from the methanogenic archaeon M. acetivorans (Ma) Archaea but also in a distantly related Gram-positive Bacterium, and the Gram-positive Bacterium D. hafniense (Dh). The gene order of the pyl indicating past horizontal gene transfer of all five genes. Here we genetic code expansion cassette is conserved, with the exception that the D. show that lateral transfer of pylTSBCD introduces biosynthesis and hafniense pylS gene homolog has been split into two genes encoding ho- genetic encoding of pyrrolysine into a naı¨veorganism. PylS-based mologs to the PylS N-terminal domain (pylSn) and the catalytic core domain assays demonstrated that pyrrolysine was biosynthesized in Esch- (pylSc) that now flank pylBCD (7).
    [Show full text]
  • Studies of the Genetic Encoding of Pyrrolysine From
    STUDIES OF THE GENETIC ENCODING OF PYRROLYSINE FROM METHANOGENIC ARCHAEA DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Anirban Mahapatra, M.Sc. ***** The Ohio State University 2007 Dissertation Committee: Dr. Joseph A. Krzycki, Advisor Approved by Dr. Juan D. Alfonzo Dr. Charles J. Daniels Dr. Kurt L. Fredrick ___________________________ Advisor, Graduate Program in Microbiology ABSTRACT Pyrrolysine is the 22nd genetically encoded amino acid to be found in nature. Co-translational insertion at in-frame UAG codons proceeds so that a single pyrrolysine residue is found in the active site of all methylamine methyltransferases required for methylamine metabolism in Methanosarcina spp. This study examines processes central to the translation of UAG as pyrrolysine. The pylT gene encoding the tRNA for pyrrolysine, tRNAPyl, is part of the pyl operon of Methanosarcina spp. which also contains the pylS gene, encoding a class II aminoacyl-tRNA synthetase; and the pylB, pylC, and pylD genes proposed to catalyze the synthesis of pyrrolysine from metabolic precursors. Here, in the first study, by characterizing a Methanosarcina acetivorans mutant with the pylT gene region deleted, the essentiality of pyrrolysine incorporation in methanogenesis from methylamines is tested. The mutant lacks detectable tRNAPyl but grows similar to wild-type on methanol or acetate for which translation of UAG as pyrrolysine is likely not to be essential. However, unlike wild-type, the mutant can not grow on any methylamine or use monomethylamine as the sole source of nitrogen. Monomethylamine methyltransferase activity is detectable in wild-type cells, but not in mutant cells during growth on methanol.
    [Show full text]
  • Improved Pyrrolysine Biosynthesis Through Phage Assisted Non-Continuous Directed Evolution of the Complete Pathway ✉ Joanne M
    ARTICLE https://doi.org/10.1038/s41467-021-24183-9 OPEN Improved pyrrolysine biosynthesis through phage assisted non-continuous directed evolution of the complete pathway ✉ Joanne M. L. Ho 1,3, Corwin A. Miller1,3, Kathryn A. Smith 1, Jacob R. Mattia1 & Matthew R. Bennett 1,2 Pyrrolysine (Pyl, O) exists in nature as the 22nd proteinogenic amino acid. Despite being a fundamental building block of proteins, studies of Pyl have been hindered by the difficulty and 1234567890():,; inefficiency of both its chemical and biological syntheses. Here, we improve Pyl biosynthesis via rational engineering and directed evolution of the entire biosynthetic pathway. To accommodate toxicity of Pyl biosynthetic genes in Escherichia coli, we also develop Alter- nating Phage Assisted Non-Continuous Evolution (Alt-PANCE) that alternates mutagenic and selective phage growths. The evolved pathway provides 32-fold improved yield of Pyl- containing reporter protein compared to the rationally engineered ancestor. Evolved PylB mutants are present at up to 4.5-fold elevated levels inside cells, and show up to 2.2-fold increased protease resistance. This study demonstrates that Alt-PANCE provides a general approach for evolving proteins exhibiting toxic side effects, and further provides an improved pathway capable of producing substantially greater quantities of Pyl-proteins in E. coli. 1 Department of Biosciences, Rice University, Houston, TX, USA. 2 Department of Bioengineering, Rice University, Houston, TX, USA. 3These authors ✉ contributed equally: Joanne M. L. Ho, Corwin A. Miller. email: [email protected] NATURE COMMUNICATIONS | (2021) 12:3914 | https://doi.org/10.1038/s41467-021-24183-9 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24183-9 yrrolysine (Pyl, O) exists in nature as the 22nd proteino- PylRS and tRNAPyl to incorporate synthetic amino acids into Pgenic amino acid1.
    [Show full text]
  • Pyrrolysine Analogues As Substrates for Pyrrolysyl-Trna Synthetase
    FEBS Letters 580 (2006) 6695–6700 Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase Carla R. Polycarpob,1, Stephanie Herringb, Ame´lie Be´rube´a,2, John L. Wooda, Dieter So¨lla,b, Alexandre Ambrogellyb,* a Department of Chemistry, Yale University, New Haven, CT 06520-8114, USA b Department of Molecular Biophysics and Biochemistry, Yale University, P.O. Box 208114, 266 Whitney Avenue, New Haven, CT 06520-8114, USA Received 24 October 2006; accepted 8 November 2006 Available online 20 November 2006 Edited by Lev Kisselev assists in the recoding event. Although putative secondary Abstract In certain methanogenic archaea a new amino acid, pyrrolysine (Pyl), is inserted at in-frame UAG codons in the structures have been identified, their role in recoding in vivo re- mRNAs of some methyltransferases. Pyl is directly acylated mains uncertain [7,8]. onto a suppressor tRNAPyl by pyrrolysyl-tRNA synthetase Determination of the crystal structure of native Methanosar- (PylRS). Due to the lack of a readily available Pyl source, we cina barkeri MtmB allowed the elucidation of the molecular looked for structural analogues that could be aminoacylated by structure of this new amino acid. Analysis of the electron PylRS onto tRNAPyl. We report here the in vitro aminoacylation density showed that pyrrolysine is a dipeptide composed of a Pyl of tRNA by PylRS with two Pyl analogues: N-e-D-prolyl-L- lysine modified at its e-N by a 4-methyl-pyrroline-5-carboxyl- lysine (D-prolyl-lysine) and N-e-cyclopentyloxycarbonyl-L-lysine Pyl ate (Fig. 1) [1]. The electron density of the MtmB crystal struc- (Cyc).
    [Show full text]
  • Carbon Source-Dependent Expansion of the Genetic Code in Bacteria
    Carbon source-dependent expansion of the genetic code in bacteria Laure Prata, Ilka U. Heinemanna, Hans R. Aernib, Jesse Rinehartb,c, Patrick O’Donoghuea,1, and Dieter Sölla,d,1 Departments of aMolecular Biophysics and Biochemistry, bCellular and Molecular Physiology, cSystems Biology Institute, and dChemistry, Yale University, New Haven, CT 06520 Contributed by Dieter Söll, October 25, 2012 (sent for review October 5, 2012) Despite the fact that the genetic code is known to vary between coli, and is sufficient to promote limited read-through of amber organisms in rare cases, it is believed that in the lifetime of a single codons (10, 11). A Methanosarcina acetivorans strain lacking cell the code is stable. We found Acetohalobium arabaticum cells tRNAPyl is viable when cells are grown on methanol as a carbon grown on pyruvate genetically encode 20 amino acids, but in the source, but detrimental for growth on methylamines (12). This presence of trimethylamine (TMA), A. arabaticum dynamically led to the initial assumption that Pyl was solely required in the expands its genetic code to 21 amino acids including pyrrolysine catalytic active site of methylamine methyltransferases that are (Pyl). A. arabaticum is the only known organism that modulates highly up-regulated on methylamines and repressed on methanol the size of its genetic code in response to its environment and (13, 14). We demonstrated, however, that Pyl can be incorporated energy source. The gene cassette pylTSBCD, required to biosynthe- in tRNAHis guanylyltransferase (Thg1), an enzyme that is not in- size and genetically encode UAG codons as Pyl, is present in the volved in methylamine metabolism, and interestingly, Pyl does not genomes of 24 anaerobic archaea and bacteria.
    [Show full text]