N-glycan chitobiose core biosynthesis by Agl24 strengthens the hypothesis of an archaeal origin of the eukaryal N-glycosylation

Protein N-glycosylation is the most common posttranslational modifications found in all three domains of life. The crenarchaeal N-glycosylation begins with the synthesis of a lipid-linked chitobiose core structure, identical to that in eukaryotes. Here, we report the identification of a thermostable archaeal beta-1,4-N-acetylglucosaminyltransferase, named archaeal glycosylation enzyme 24 (Agl24), responsible for the synthesis of the N-glycan chitobiose core. Biochemical characterization confirmed the function as an inverting β-D-GlcNAc-(1→4)-α-D-GlcNAc-diphosphodolichol glycosyltransferase. Substitution of a conserved histidine residue, found also in the eukaryotic and bacterial homologs, demonstrated its functional importance for Agl24. Furthermore, bioinformatics and structural modeling revealed strong similarities between Agl24 and both the eukaryotic Alg14/13 and a distant relation to the bacterial MurG, which catalyze the identical or a similar process, respectively. Our data, complemented by phylogenetic analysis of Alg13 and Alg14, revealed similar sequences in Asgardarchaeota, further supporting the hypothesis that the Alg13/14 homologs in eukaryotes have been acquired during eukaryogenesis.HighlightsFirst identification and characterization of a thermostable β-D-GlcNAc-(1→4)-α-D-GlcNAc-diphosphodolichol glycosyltransferase (GT family 28) in Archaea.A highly conserved histidine, within a GGH motif in Agl24, Alg14, and MurG, is essential for function of Agl24.Agl24-like homologs are broadly distributed among Archaea.The eukaryotic Alg13 and Alg14 are closely related to the Asgard homologs, suggesting their acquisition during eukaryogenesis.

Novel ribonucleotide discrimination in the RNA polymerase-like two-barrel catalytic core of Family D DNA polymerases

Family D DNA polymerase (PolD) is the essential replicative DNA polymerase for duplication of most archaeal genomes. PolD contains a unique two-barrel catalytic core absent from all other DNA polymerase families but found in RNA polymerases (RNAPs). While PolD has an ancestral RNA polymerase catalytic core, its active site has evolved the ability to discriminate against ribonucleotides. Until now, the mechanism evolved by PolD to prevent ribonucleotide incorporation was unknown. In all other DNA polymerase families, an active site steric gate residue prevents ribonucleotide incorporation. In this work, we identify two consensus active site acidic (a) and basic (b) motifs shared across the entire two-barrel nucleotide polymerase superfamily, and a nucleotide selectivity (s) motif specific to PolD versus RNAPs. A novel steric gate histidine residue (H931 in Thermococcus sp. 9°N PolD) in the PolD s-motif both prevents ribonucleotide incorporation and promotes efficient dNTP incorporation. Further, a PolD H931A steric gate mutant abolishes ribonucleotide discrimination and readily incorporates a variety of 2′ modified nucleotides. Taken together, we construct the first putative nucleotide bound PolD active site model and provide structural and functional evidence for the emergence of DNA replication through the evolution of an ancestral RNAP two-barrel catalytic core.

α2-Macroglobulin-like protein 1 can conjugate and inhibit proteases through their hydroxyl groups, because of an enhanced reactivity of its thiol ester

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α2-macroglobulin–like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity–conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment.

Promiscuous DNA cleavage by HpyAII endonuclease is modulated by the HNH catalytic residues

Helicobacter pylori is a carcinogenic bacterium that is responsible for 5.5% of all human gastric cancers. H. pylori codes for an unusually large number of restriction–modification (R–M) systems and several of them are strain-specific and phase-variable. HpyAII is a novel Type IIs phase-variable restriction endonuclease present in 26695 strain of H. pylori. We show that HpyAII prefers two-site substrates over one-site substrates for maximal cleavage activity. HpyAII is less stringent in metal ion requirement and shows higher cleavage activity with Ni2+ over Mg2+. Mutational analysis of the putative residues of the HNH motif of HpyAII confirms that the protein has an active HNH site for the cleavage of DNA. However, mutation of the first Histidine residue of the HNH motif to Alanine does not abolish the enzymatic activity, but instead causes loss of fidelity compared with wildtype HpyAII. Previous studies have shown that mutation of the first Histidine residue of the HNH motif of all other known HNH motif motif-containing enzymes completely abolishes enzymatic activity. We found, in the case of HpyAII, mutation of an active site residue leads to the loss of endonuclease fidelity. The present study provides further insights into the evolution of restriction enzymes.

Histidine Protonation Controls Structural Heterogeneity in the Cyanobacteriochrome AnPixJg2

Cyanobacteriochromes are compact and spectrally diverse photoreceptor proteins that bind a linear tetrapyrrole as a chromophore. They show photochromicity by having two stable states that can be interconverted by the photoisomerization of the chromophore. Hence, these photochemical properties make them an attractive target for biotechnological applications. However, their application is impeded by structural heterogeneity that reduces the yield of the photoconversion. The heterogeneity can originate either from the chromophore structure or the protein environment. Here, we study the origin of the heterogeneity in AnPixJg2, a representative member of the red/green cyanobacteriochrome family, that has a red absorbing parental state and a green absorbing photoproduct state. Using molecular dynamics simulations and umbrella sampling we have identified the protonation state of a conserved histidine residue as a trigger for structural heterogeneity. When the histidine is in a neutral form, the chromophore structure is homogenous, while in a positively charged form, the chromophore is heterogeneous with two different conformations. We have identified a correlation between the protonation of the histidine and the structural heterogeneity of the chromophore by detailed characterization of the interactions in the protein binding site. Our findings reconcile seemingly contradicting spectroscopic studies that attribute the heterogeneity to different sources. Furthermore, we predict that circular dichroism can be used as a diagnostic tool to distinguish different substates.Significance statementCyanobacteriochromes are photoreceptor proteins that have attracted attention for their immense potential in bioimaging and optogenetics applications. This is due to their desirable properties such as compactness, photochromicity and diverse spectral tuning. Despite these advantages, nature has set a limitation in the form of structural heterogeneity that presents a drawback for its application in biotechnology. We have identified a histidine residue in the vicinity of the chromophore as the origin of the heterogeneity in red/green CBCRs. The protonation state of this conserved histidine alters an extended network of protein-chromophore interactions and induces heterogeneity. Furthermore, theoretical CD spectroscopy has revealed easy identification of heterogeneity. Hence, our study paves the way for rational design and optimization of protein properties.

Tartryl-CoA inhibits succinyl-CoA synthetase

Succinyl-CoA synthetase (SCS) catalyzes the only substrate-level phosphorylation step in the tricarboxylic acid cycle. Human GTP-specific SCS (GTPSCS), an αβ-heterodimer, was produced in Escherichia coli. The purified protein crystallized from a solution containing tartrate, CoA and magnesium chloride, and a crystal diffracted to 1.52 Å resolution. Tartryl-CoA was discovered to be bound to GTPSCS. The CoA portion lies in the amino-terminal domain of the α-subunit and the tartryl end extends towards the catalytic histidine residue. The terminal carboxylate binds to the phosphate-binding site of GTPSCS.

Neutron crystallography of copper amine oxidase reveals keto/enolate interconversion of the quinone cofactor and unusual proton sharing

Recent advances in neutron crystallographic studies have provided structural bases for quantum behaviors of protons observed in enzymatic reactions. Thus, we resolved the neutron crystal structure of a bacterial copper (Cu) amine oxidase (CAO), which contains a prosthetic Cu ion and a protein-derived redox cofactor, topa quinone (TPQ). We solved hitherto unknown structures of the active site, including a keto/enolate equilibrium of the cofactor with a nonplanar quinone ring, unusual proton sharing between the cofactor and the catalytic base, and metal-induced deprotonation of a histidine residue that coordinates to the Cu. Our findings show a refined active-site structure that gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions.