Codon optimality is the primary contributor to the exceptional mutational sensitivity of CcdA antitoxin in its operonic context

Deep mutational scanning studies suggest that single synonymous mutations are typically silent and that most exposed, non active-site residues are tolerant to mutations. Here we show that the ccdA antitoxin component of the E.coli ccdAB toxin-antitoxin operonic system is unusually sensitive to mutations when studied in the operonic context. A large fraction (~80%) of single codon mutations, including many synonymous mutations in the ccdA gene show inactive phenotypes that are correlated with the E.coli codon usage frequency but retain native-like binding affinity towards cognate toxin, CcdB. Therefore, the observed phenotypic effects are largely not due to alterations in protein structure or stability, consistent with the fact that a large region of CcdA is intrinsically disordered. In select cases, proteomics studies reveal altered ratios of CcdA:CcdB protein levels in vivo, suggesting that these mutations likely alter relative translation efficiencies of the two genes in the operon. We extend these results by predicting and validating single synonymous mutations that lead to loss of function phenotypes in the relBE operon upon introduction of rarer codons. Thus, in their native context, genes are likely to be more sensitive to both synonymous and non-synonymous point mutations than inferred from previous saturation mutagenesis studies.

Structure of UBE2K–Ub/E3/polyUb reveals mechanisms of K48-linked Ub chain extension

Ubiquitin (Ub) chain types govern distinct biological processes. K48-linked polyUb chains target substrates for proteasomal degradation, but the mechanism of Ub chain synthesis remains elusive due to the transient nature of Ub handover. Here, we present the structure of a chemically trapped complex of the E2 UBE2K covalently linked to donor Ub and acceptor K48-linked di-Ub, primed for K48-linked Ub chain synthesis by a RING E3. The structure reveals the basis for acceptor Ub recognition by UBE2K active site residues and the C-terminal Ub-associated (UBA) domain, to impart K48-linked Ub specificity and catalysis. Furthermore, the structure unveils multiple Ub-binding surfaces on the UBA domain that allow distinct binding modes for K48- and K63-linked Ub chains. This multivalent Ub-binding feature serves to recruit UBE2K to ubiquitinated substrates to overcome weak acceptor Ub affinity and thereby promote chain elongation. These findings elucidate the mechanism of processive K48-linked polyUb chain formation by UBE2K.

Plant peptidoglycan precursor biosynthesis: Conservation between moss chloroplasts and Gram negative bacteria

An accumulation of evidence suggests that peptidoglycan, consistent with a bacterial cell wall, is synthesised around the chloroplasts of many photosynthetic eukaryotes, from glaucophyte algae to land plants at least as evolved as pteridophyte ferns, but the biosynthetic pathway has not been demonstrated. We employed mass spectrometry and enzymology in a two fold approach to characterize the synthesis of peptidoglycan in chloroplasts of the moss Physcomitrium (Physcomitrella) patens. To drive the accumulation of peptidoglycan pathway intermediates, P.patens was cultured with the antibiotics phosphomycin, D-cycloserine and carbenicillin, which inhibit key peptidoglycan pathway proteins in bacteria. Mass spectrometry of the TCA-extracted moss metabolome revealed elevated levels of five of the predicted intermediates from UDP-GlcNAc through to the UDP-MurNAc-D,L-diaminopimelate (DAP)-pentapeptide. Most Gram negative bacteria, including cyanobacteria, incorporate meso-diaminopimelate (D,L-DAP) into the third residue of the stem peptide of peptidoglycan, as opposed to L-Lysine, typical of most Gram positive bacteria. To establish the specificity of D,L-DAP incorporation into the P.patens precursors, we analysed the recombinant protein that appends the third stem peptide amino acid, UDP-MurNAc-tripeptide ligase (MurE), from both P.patens and the cyanobacterium Anabaena sp. strain PCC 7120. Both ligases incorporated D,L-DAP in almost complete preference to L-Lys, consistent with the mass spectrophotometric data, with catalytic efficiencies similar to previously documented Gram negative bacterial MurE ligases. We discuss how these data accord with the conservation of active site residues common to DL-DAP-incorporating bacterial MurE ligases and of the probability of a horizontal gene transfer event within the plant peptidoglycan pathway.

Highly Stable, Cold-Active Aldehyde Dehydrogenase from the Marine Antarctic Flavobacterium sp. PL002

Stable aldehyde dehydrogenases (ALDH) from extremophilic microorganisms constitute efficient catalysts in biotechnologies. In search of active ALDHs at low temperatures and of these enzymes from cold-adapted microorganisms, we cloned and characterized a novel recombinant ALDH from the psychrotrophic Flavobacterium PL002 isolated from Antarctic seawater. The recombinant enzyme (F-ALDH) from this cold-adapted strain was obtained by cloning and expressing of the PL002 aldH gene (1506 bp) in Escherichia coli BL21(DE3). Phylogeny and structural analyses showed a high amino acid sequence identity (89%) with Flavobacterium frigidimaris ALDH and conservation of all active site residues. The purified F-ALDH by affinity chromatography was homotetrameric, preserving 80% activity at 4 °C for 18 days. F-ALDH used both NAD+ and NADP+ and a broad range of aliphatic and aromatic substrates, showing cofactor-dependent compensatory KM and kcat values and the highest catalytic efficiency (0.50 µM−1 s−1) for isovaleraldehyde. The enzyme was active in the 4–60 °C-temperature interval, with an optimal pH of 9.5, and a preference for NAD+-dependent reactions. Arrhenius plots of both NAD(P)+-dependent reactions indicated conformational changes occurring at 30 °C, with four(five)-fold lower activation energy at high temperatures. The high thermal stability and substrate-specific catalytic efficiency of this novel cold-active ALDH favoring aliphatic catalysis provided a promising catalyst for biotechnological and biosensing applications.

Evolution of tunnels in alpha/beta-hydrolase fold proteins - what can we learn from studying epoxide hydrolases?

The evolutionary variability of a protein's residues is highly dependent on protein region and protein function. Solvent-exposed residues, excluding those at interaction interfaces, are more variable than buried residues. Active site residues are considered to be conserved as they ensure an enzyme's activity and selectivity. The abovementioned rules apply also to α/β-hydrolase fold proteins - an example of enzymes with buried active sites equipped with tunnels linking the reaction site with the exterior. We hypothesised two scenarios: (1) tunnels are lined by mostly variable residues, allowing adaptation to the evolutionary pressures of a changeable environment; or (2) tunnels are lined by mostly conserved amino acids, and are equipped with a number of specific variable residues that are able to respond to evolutionary pressure. We also wanted to check if evolutionary analysis can help distinguish functional and non-functional tunnels. Soluble epoxide hydrolases (sEHs) represent a good case study for the analysis of the evolution of tunnels in an α/β-hydrolase fold family due to their size and architecture. Here, we propose methods for the comparison of tunnels detected in both crystal structures and molecular dynamics simulations, as well as the assignment of tunnel functionality, and we identify critical steps for careful tunnel inspection. We also compare the entropy values of the tunnel-lining residues and system-specific compartments in seven selected sEHs from different clades. We present three different cases of entropy distribution among tunnel-lining residues. As a result, we propose a 'perforation' model for tunnel evolution via the merging of internal cavities or surface perforations. We also report an approach for the identification of highly variable tunnel-lining residues as potential targets to be used for the fine-tuning of selected enzymes.

Towards Computationally Guided Design and Engineering of a Neisseria meningitidis Serogroup W Capsule Polymerase with Altered Substrate Specificity

Heavy metal contamination of drinking water is a public health concern that requires the development of more efficient bioremediation techniques. Absorption technologies, including biosorption, provide opportunities for improvements to increase the diversity of target metal ions and overall binding capacity. Microorganisms are a key component in wastewater treatment plants, and they naturally bind metal ions through surface macromolecules but with limited capacity. The long-term goal of this work is to engineer capsule polymerases to synthesize molecules with novel functionalities. In previously published work, we showed that the Neisseria meningitidis serogroup W (NmW) galactose–sialic acid (Gal–NeuNAc) heteropolysaccharide binds lead ions effectively, thereby demonstrating the potential for its use in environmental decontamination applications. In this study, computational analysis of the NmW capsule polymerase galactosyltransferase (GT) domain was used to gain insight into how the enzyme could be modified to enable the synthesis of N-acetylgalactosamine–sialic acid (GalNAc–NeuNAc) heteropolysaccharide. Various computational approaches, including molecular modeling with I-TASSER and molecular dynamics (MD) simulations with NAMD, were utilized to identify key amino acid residues in the substrate binding pocket of the GT domain that may be key to conferring UDP-GalNAc specificity. Through these combined strategies and using BshA, a UDP-GlcNAc transferase, as a structural template, several NmW active site residues were identified as mutational targets to accommodate the proposed N-acetyl group in UDP-GalNAc. Thus, a rational approach for potentially conferring new properties to bacterial capsular polysaccharides is demonstrated.

Identification of the active site and characterization of a novel sporulation-specific cysteine protease YabG from Bacillus subtilis

In order to characterize the probable protease gene yabG found in the genomes of spore-forming bacteria, Bacillus subtilis yabG was expressed as a 35 kDa His-tagged protein (BsYabG) in Escherichia coli cells. During purification using Ni-affinity chromatography, the 35 kDa protein was degraded via several intermediates to form a 24 kDa protein. Furthermore, it was degraded after an extended incubation period. The effect of protease inhibitors, including certain chemical modification reagents, on the conversion of the 35 kDa protein to the 24 kDa protein was investigated. Reagents reacting with sulfhydryl groups exerted significant effects, strongly suggesting that the yabG gene product is a cysteine protease with autolytic activity. Site-directed mutagenesis of the conserved Cys and His residues indicated that Cys218 and His172 are active site residues. No degradation was observed in the C218A/S and H172A mutants. In addition to the chemical modification reagents, benzamidine inhibited the degradation of the 24 kDa protein. Determination of the N-terminal amino acid sequences of the intermediates revealed trypsin-like specificity for YabG protease. Based on the relative positions of His172 and Cys218 and their surrounding sequences, we propose the classification of YabG as a new family of clan CD in the Merops peptidase database.

Isoliquiritigenin, a potent human monoamine oxidase inhibitor, modulates dopamine D1, D3, and vasopressin V1A receptors

Isoliquiritigenin (= 4,2′,4′-Trihydroxychalcone) (ILG) is a major constituent of the Glycyrrhizae Rhizoma that has significant neuroprotective functions. In the present study, we re-examined the potential of ILG to inhibit human monoamine oxidase (hMAO) in vitro and established its mechanism of inhibition through a kinetics study and molecular docking examination. ILG showed competitive inhibition of hMAO-A and mixed inhibition of hMAO-B with IC50 values of 0.68 and 0.33 µM, respectively, which varied slightly from the reported IC50 values. Since ILG has been reported to reduce dopaminergic neurodegeneration and psychostimulant-induced toxicity (both of which are related to dopamine and vasopressin receptors), we investigated the binding affinity and modulatory functions of ILG on dopamine and vasopressin receptors. ILG was explored as an antagonist of the D1 receptor and an agonist of the D3 and V1A receptors with good potency. An in silico docking investigation revealed that ILG can interact with active site residues at target receptors with low binding energies. These activities of ILG on hMAO and brain receptors suggest the potential role of the compound to ameliorate dopaminergic deficits, depression, anxiety, and associated symptoms in Parkinson’s disease and other neuronal disorders.

Structural analysis of the sulfatase AmAS from Akkermansia muciniphila

Akkermansia muciniphila, an anaerobic Gram-negative bacterium, is a major intestinal commensal bacterium that can modulate the host immune response. It colonizes the mucosal layer and produces nutrients for the gut mucosa and other commensal bacteria. It is believed that mucin desulfation is the rate-limiting step in the mucin-degradation process, and bacterial sulfatases that carry out mucin desulfation have been well studied. However, little is known about the structural characteristics of A. muciniphila sulfatases. Here, the crystal structure of the premature form of the A. muciniphila sulfatase AmAS was determined. Structural analysis combined with docking experiments defined the critical active-site residues that are responsible for catalysis. The loop regions I–V were proposed to be essential for substrate binding. Structure-based sequence alignment and structural superposition allow further elucidation of how different subclasses of formylglycine-dependent sulfatases (FGly sulfatases) adopt the same catalytic mechanism but exhibit diverse substrate specificities. These results advance the understanding of the substrate-recognition mechanisms of A. muciniphila FGly-type sulfatases. Structural variations around the active sites account for the different substrate-binding properties. These results will enhance the understanding of the roles of bacterial sulfatases in the metabolism of glycans and host–microbe interactions in the human gut environment.

Improving the Stability and Activity of Arginine Decarboxylase at Alkaline pH for the Production of Agmatine

Agmatine, involved in various modulatory actions in cellular mechanisms, is produced from arginine (Arg) by decarboxylation reaction using arginine decarboxylase (ADC, EC 4.1.1.19). The major obstacle of using wild-type Escherichia coli ADC (ADCes) in agmatine production is its sharp activity loss and instability at alkaline pH. Here, to overcome this problem, a new disulfide bond was rationally introduced in the decameric interface region of the enzyme. Among the mutants generated, W16C/D43C increased both thermostability and activity. The half-life (T1/2) of W16C/D43C at pH 8.0 and 60°C was 560 min, which was 280-fold longer than that of the wild-type, and the specific activity at pH 8.0 also increased 2.1-fold. Site-saturation mutagenesis was subsequently performed at the active site residues of ADCes using the disulfide-bond mutant (W16C/D43C) as a template. The best variant W16C/D43C/I258A displayed a 4.4-fold increase in the catalytic efficiency when compared with the wild-type. The final mutant (W16C/D43C/I258A) was successfully applied to in vitro synthesis of agmatine with an improved yield and productivity (>89.0% yield based on 100 mM of Arg within 5 h).