New Processes for Ionizing Nonvolatile Compounds in Mass Spectrometry: The Road of Discovery to Current State-of-the-Art.

This covers discovery and mechanistic aspects as well as initial applications of novel ionization processes for use in mass spectrometry that guided us in a series of subsequent discoveries, instrument developments, and commercialization. matrix-assisted ionization on an intermediate pressure matrix-assisted laser desorption/ionization source the use of a laser, high voltages, or any other added energy was simply unbelievable, at first. Individually and as a whole, the various discoveries and inventions started to paint, , an exciting new picture and outlook in mass spectrometry from which key developments grew that were at the time unimaginable, and continue to surprise us in its simplistic preeminence.

A Soluble, Minimalistic Glycosylphosphatidylinositol Transamidase (GPI-T) Retains Transamidation Activity.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a eukaryotic, post-translational modification catalyzed by GPI transamidase (GPI-T). The GPI-T is composed of five membrane-bound subunits: Gpi8, Gaa1, Gpi16, Gpi17, and Gab1. GPI-T has been recalcitrant to structure and function studies because of its complexity and membrane-solubility.

Computed structures of core eukaryotic protein complexes.

Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take advantage of advances in proteome-wide amino acid coevolution analysis and deep-learning–based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes within the proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.

Clinically Relevant Mutations of Mycobacterial GatCAB Inform Regulation of Translational Fidelity.

Most bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNA and tRNA. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction in the second step of this pathway. We have previously demonstrated in mycobacteria that this two-step pathway is error prone and translational errors contribute to adaptive phenotypes such as antibiotic tolerance.

Did Amino Acid Side Chain Reactivity Dictate the Composition and Timing of Aminoacyl-tRNA Synthetase Evolution?

The twenty amino acids in the standard genetic code were fixed prior to the last universal common ancestor (LUCA). Factors that guided this selection included establishment of pathways for their metabolic synthesis and the concomitant fixation of substrate specificities in the emerging aminoacyl-tRNA synthetases (aaRSs). In this conceptual paper, we propose that the chemical reactivity of some amino acid side chains (e.

Anticodon-binding domain swapping in a nondiscriminating aspartyl-tRNA synthetase reveals contributions to tRNA specificity and catalytic activity.

The nondiscriminating aspartyl-tRNA synthetase (ND-AspRS), found in many archaea and bacteria, covalently attaches aspartic acid to tRNA and tRNA generating a correctly charged Asp-tRNA and an erroneous Asp-tRNA . This relaxed tRNA specificity is governed by interactions between the tRNA and the enzyme. In an effort to assess the contributions of the anticodon-binding domain to tRNA specificity, we constructed two chimeric enzymes, Chimera-D and Chimera-N, by replacing the native anticodon-binding domain in the Helicobacter pylori ND-AspRS with that of a discriminating AspRS (Chimera-D) and an asparaginyl-tRNA synthetase (AsnRS, Chimera-N), both from Escherichia coli.

Putting amino acids onto tRNAs: The aminoacyl-tRNA synthetases as catalysts.

In this chapter we consider the catalytic approaches used by aminoacyl-tRNA synthetase (AARS) enzymes to synthesize aminoacyl-tRNA from cognate amino acid and tRNA. This ligase reaction proceeds through an activated aminoacyl-adenylate (aa-AMP). Common themes among AARSs include use of induced fit to drive catalysis and transition state stabilization by class-conserved sequence and structure motifs.

Bacterial Aspartyl-tRNA Synthetase Has Glutamyl-tRNA Synthetase Activity.

The aminoacyl-tRNA synthetases (aaRSs) are well established as the translators of the genetic code, because their products, the aminoacyl-tRNAs, read codons to translate messenger RNAs into proteins. Consequently, deleterious errors by the aaRSs can be transferred into the proteome via misacylated tRNAs. Nevertheless, many microorganisms use an indirect pathway to produce Asn-tRNA via Asp-tRNA.

Old enzymes new herbicides.

The introduction of manmade chemicals, including the herbicide atrazine, into the environment has led to the emergence of microorganisms with new biodegradation pathways. Esquirol demonstrate that the AtzE enzyme catalyzes a central step in atrazine degradation and that expression of AtzE requires coexpression of the small protein AtzG. Remarkably, AtzG and AtzE appear to have evolved from GatC and GatA, components of an ancient enzyme involved in indirect tRNA aminoacylation, providing an elegant demonstration of metabolic repurposing.

Indirect tRNA aminoacylation during accurate translation and phenotypic mistranslation.

The fact that most bacteria do not contain a full set of aminoacyl-tRNA synthetases (aaRS) is often underappreciated. In the absence of asparaginyl-tRNA and/or glutaminyl-tRNA synthetase (AsnRS and GlnRS), Asn-tRNA and/or Gln-tRNA are produced by an indirect tRNA aminoacylation pathway that relies on misacylation of these two tRNAs by two different misacylating aaRSs, followed by transamidation by an amidotransferase (GatCAB in bacteria). This review highlights the central importance of indirect tRNA aminoacylation to accurate protein translation, mechanistic peculiarities that appear to be unique to this system, and the newly recognized connection between indirect tRNA aminoacylation and mistranslation as a strategy used by bacteria to respond to environmental stressors like antibiotics.

The soluble domains of Gpi8 and Gaa1, two subunits of glycosylphosphatidylinositol transamidase (GPI-T), assemble into a complex.

Glycosylphosphatidylinositol transamidase (GPI-T) catalyzes the post-translational addition of the GPI anchor to the C-terminus of some proteins. In most eukaryotes, Gpi8, the active site subunit of GPI-T, is part of a hetero-pentameric complex containing Gpi16, Gaa1, Gpi17, and Gab1. Gpi8, Gaa1, and Gpi16 co-purify as a heterotrimer from Saccharomyces cerevisiae, suggesting that they form the core of the GPI-T.

OB or Not OB: Idiosyncratic utilization of the tRNA-binding OB-fold domain in unicellular, pathogenic eukaryotes.

In this review, we examine the so-called OB-fold, a tRNA-binding domain homologous to the bacterial tRNA-binding protein Trbp111. We highlight the ability of OB-fold homologs to bind tRNA species and summarize their distribution in evolution. Nature has capitalized on the advantageous effects acquired when an OB-fold domain binds to tRNA by evolutionarily selecting this domain for fusion to different enzymes.

Characterization of tunnel mutants reveals a catalytic step in ammonia delivery by an aminoacyl-tRNA amidotransferase.

The Helicobacter pylori Asp-tRNA(A) (sn) /Glu-tRNA(G) (ln) amidotransferase (GatCAB) utilizes an uncommonly hydrophilic, ~ 40 Å ammonia tunnel for ammonia/ammonium transport between isolated active sites. Hydrophilicity of this tunnel requires a distinct ammonia transport mechanism, which hypothetically occurs through a series of deprotonation and protonation steps. To explore the initiation of this relay mechanism, the highly conserved tunnel residue D185 (in the GatA subunit) was enzymatically and computationally investigated by comparing D185A, D185N, and D185E mutant enzymes to wild-type GatCAB.

Integrating responsible conduct of research education into undergraduate biochemistry and molecular biology laboratory curricula.

Recently, a requirement for directed responsible conduct in research (RCR) education has become a priority in the United States and elsewhere. In the US, both the National Institutes of Health and the National Science Foundation require RCR education for all students who are financially supported by federal awards. The guidelines produced by these agencies offer useful templates for the introduction of RCR materials into courses worldwide.

GPI transamidase and GPI anchored proteins: oncogenes and biomarkers for cancer.

Cancer is second only to heart disease as a cause of death in the US, with a further negative economic impact on society. Over the past decade, details have emerged which suggest that different glycosylphosphatidylinositol (GPI)-anchored proteins are fundamentally involved in a range of cancers. This post-translational glycolipid modification is introduced into proteins via the action of the enzyme GPI transamidase (GPI-T).

A tRNA-independent mechanism for transamidosome assembly promotes aminoacyl-tRNA transamidation.

Many bacteria lack genes encoding asparaginyl- and/or glutaminyl-tRNA synthetase and consequently rely on an indirect path for the synthesis of both Asn-tRNA(Asn) and Gln-tRNA(Gln). In some bacteria such as Thermus thermophilus, efficient delivery of misacylated tRNA to the downstream amidotransferase (AdT) is ensured by formation of a stable, tRNA-dependent macromolecular complex called the Asn-transamidosome. This complex enables direct delivery of Asp-tRNA(Asn) from the non-discriminating aspartyl-tRNA synthetase to AdT, where it is converted into Asn-tRNA(Asn).

Defining the boundaries of species specificity for the Saccharomyces cerevisiae glycosylphosphatidylinositol transamidase using a quantitative in vivo assay.

In eukaryotes, GPI (glycosylphosphatidylinositol) lipid anchoring of proteins is an abundant post-translational modification. The attachment of the GPI anchor is mediated by GPI-T (GPI transamidase), a multimeric, membrane-bound enzyme located in the ER (endoplasmic reticulum). Upon modification, GPI-anchored proteins enter the secretory pathway and ultimately become tethered to the cell surface by association with the plasma membrane and, in yeast, by covalent attachment to the outer glucan layer.

The asparagine-transamidosome from Helicobacter pylori: a dual-kinetic mode in non-discriminating aspartyl-tRNA synthetase safeguards the genetic code.

Helicobacter pylori catalyzes Asn-tRNA(Asn) formation by use of the indirect pathway that involves charging of Asp onto tRNA(Asn) by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS), followed by conversion of the mischarged Asp into Asn by the GatCAB amidotransferase. We show that the partners of asparaginylation assemble into a dynamic Asn-transamidosome, which uses a different strategy than the Gln-transamidosome to prevent the release of the mischarged aminoacyl-tRNA intermediate. The complex is described by gel-filtration, dynamic light scattering and kinetic measurements.

The kinase activity of the Helicobacter pylori Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase is sensitive to distal mutations in its putative ammonia tunnel.

The Helicobacter pylori (Hp) Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase (AdT) plays important roles in indirect aminoacylation and translational fidelity. AdT has two active sites, in two separate subunits. Kinetic studies have suggested that interdomain communication occurs between these subunits; however, this mechanism is not well understood.

Gln-tRNAGln synthesis in a dynamic transamidosome from Helicobacter pylori, where GluRS2 hydrolyzes excess Glu-tRNAGln.

In many bacteria and archaea, an ancestral pathway is used where asparagine and glutamine are formed from their acidic precursors while covalently linked to tRNA(Asn) and tRNA(Gln), respectively. Stable complexes formed by the enzymes of these indirect tRNA aminoacylation pathways are found in several thermophilic organisms, and are called transamidosomes. We describe here a transamidosome forming Gln-tRNA(Gln) in Helicobacter pylori, an ε-proteobacterium pathogenic for humans; this transamidosome displays novel properties that may be characteristic of mesophilic organisms.

Synthesis of truncated analogues for studying the process of glycosyl phosphatidylinositol modification.

Many eukaryotic proteins are modified with a glycosylphosphatidylinositol (GPI) anchor at their C-termini. This post-translational modification causes these proteins to be noncovalently tethered to the plasma membrane. The synthesis of truncated GPI anchor analogues is reported; these compounds were designed for use as soluble substrates for GPI transamidase (GPI-T), the enzyme that appends the GPI anchor onto proteins.

Recognition of tRNAGln by Helicobacter pylori GluRS2--a tRNAGln-specific glutamyl-tRNA synthetase.

Accurate aminoacylation of tRNAs by the aminoacyl-tRNA synthetases (aaRSs) plays a critical role in protein translation. However, some of the aaRSs are missing in many microorganisms. Helicobacter pylori does not have a glutaminyl-tRNA synthetase (GlnRS) but has two divergent glutamyl-tRNA synthetases: GluRS1 and GluRS2.