Roles of active site residues in catalysis, substrate binding, cooperativity and the reaction mechanism of the quinoprotein glycine oxidase [Enzymology]

March 31st, 2020 by Kyle J. Mamounis, Erik T Yukl, Victor L. Davidson

The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetremeric enzyme exhibits strong cooperativity towards glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766 and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on kcat and K0.5 for catalysis, K0.5 for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.
  • Posted in Journal of Biological Chemistry, Publications
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The single CCA-adding enzyme of T. brucei has distinct functions in the cytosol and in mitochondria [RNA]

March 31st, 2020 by Shikha Shikha, Andre Schneider

tRNAs universally carry a CCA nucleotide triplet at their 3′-ends. In eukaryotes, the CCA is added post-transcriptionally by the CCA-adding enzyme (CAE). The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes and therefore imports all of its tRNAs from the cytosol. This has generated interest in the tRNA modifications and their distribution in this organism, including how CCA is added to tRNAs. Here, using a BLAST search for genes encoding putative CAE proteins in T. brucei, we identified a single ORF, Tb927.9.8780, as a potential candidate. Knockdown of this putative protein, termed TbCAE, resulted in the accumulation of truncated tRNAs, abolished translation, and inhibited both total and mitochondrial CCA-adding activities, indicating that TbCAE is located both in the cytosol and mitochondrion. However, mitochondrially localized tRNAs were much less affected by the TbCAE ablation than the other tRNAs. Complementation assays revealed that the N-terminal 10 amino acids of TbCAE are dispensable for its activity and mitochondrial localization and that deletion of 10 further amino acids abolishes both. A growth arrest caused by the TbCAE knockdown was rescued by the expression of the cytosolic isoform of yeast CAE, even though it was not imported into mitochondria. This finding indicated that the yeast enzyme complements the essential function of TbCAE by adding CCA to the primary tRNA transcripts. Of note, ablation of the mitochondrial TbCAE activity, which likely has a repair function, only marginally affected growth.

An aminoacylation ribozyme evolved from a natural tRNA-sensing T-box riboswitch

March 23rd, 2020 by Satoshi Ishida

Nature Chemical Biology, Published online: 23 March 2020; doi:10.1038/s41589-020-0500-6

The authors develop a ribozyme, Tx2.1, that is capable of aminoacylating tRNA with specificity for the anticodon from directed evolution of a T-box riboswitch. Tx2.1 could be used to charge non-natural amino acids in an in vitro translation system.
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The origin of genomic <i>N</i><sup>6</sup>-methyl-deoxyadenosine in mammalian cells

March 23rd, 2020 by Michael U. Musheev

Nature Chemical Biology, Published online: 23 March 2020; doi:10.1038/s41589-020-0504-2

A metabolic labeling method reveals that genomic N6-methyl-deoxyadenosine in mammalian cell lines originates not from direct methylation in DNA, but from a misincorporation of the metabolite of ribo-N6-methyladenosine.
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A gut reaction

March 20th, 2020 by Mirella Bucci

Nature Chemical Biology, Published online: 20 March 2020; doi:10.1038/s41589-020-0514-0

A gut reaction

Locked on target

March 20th, 2020 by Caitlin Deane

Nature Chemical Biology, Published online: 20 March 2020; doi:10.1038/s41589-020-0512-2

Locked on target

Unifying principles of bifunctional, proximity-inducing small molecules

March 20th, 2020 by Christopher J. Gerry

Nature Chemical Biology, Published online: 20 March 2020; doi:10.1038/s41589-020-0469-1

This Perspective describes the chemical and biophysical principles common to all bifunctional, proximity-inducing small molecules. It also discusses the underappreciated diversity of their chemical structures and biological mechanisms.
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Tick tock

March 20th, 2020 by Yiyun Song

Nature Chemical Biology, Published online: 20 March 2020; doi:10.1038/s41589-020-0515-z

Tick tock

A tell-tale absence

March 20th, 2020 by Caitlin Deane

Nature Chemical Biology, Published online: 20 March 2020; doi:10.1038/s41589-020-0513-1

A tell-tale absence

A new path to tyrosine sulfation

March 16th, 2020 by Chang C. Liu

Nature Chemical Biology, Published online: 16 March 2020; doi:10.1038/s41589-020-0482-4

Tyrosine sulfation is a common post-translational modification known to play critical roles in multiple bioprocesses. A cleverly engineered mammalian expanded genetic code now enables the direct co-translational incorporation of tyrosine sulfates into proteins to study their function in cellular contexts.