De Novo Resistance to Arg10-Teixobactin Occurs Slowly and Is Costly.

Bacterial pathogens are rapidly evolving resistance to all clinically available antibiotics. One part of the solution to this complex issue is to better understand the resistance mechanisms to new and existing antibiotics. Here, we focus on two antibiotics. Teixobactin is a recently discovered promising antibiotic that is claimed to "kill pathogens without detectable resistance" (L. L. Ling, T. Schneider, A. J. Peoples, A. L. Spoering, et al., Nature 517:455-459, 2015, https://doi.org/10.1038/nature14098). Moenomycin A has been extensively used in animal husbandry for over 50 years with no meaningful antibiotic resistance arising. However, the nature, mechanisms, and consequences of the evolution of resistance to these "resistance-proof" compounds have not been investigated. Through a fusion of experimental evolution, whole-genome sequencing, and structural biology, we show that Staphylococcus aureus can develop significant resistance to both antibiotics in clinically meaningful timescales. The magnitude of evolved resistance to Arg10-teixobactin is 300-fold less than to moenomycin A over 45 days, and these are 2,500-fold and 8-fold less than evolved resistance to rifampicin (control), respectively. We have identified a core suite of key mutations, which correlate with the evolution of resistance, that are in genes involved in cell wall modulation, lipid synthesis, and energy metabolism. We show the evolution of resistance to these antimicrobials translates into significant cross-resistance against other clinically relevant antibiotics for moenomycin A but not Arg10-teixobactin. Lastly, we show that resistance is rapidly lost in the absence of antibiotic selection, especially for Arg10-teixobactin. These findings indicate that teixobactin is worth pursuing for clinical applications and provide evidence to inform strategies for future compound development and clinical management.

The C-Type Lysozyme from the upper Gastrointestinal Tract of Opisthocomus hoatzin, the Stinkbird.

Muramidases/lysozymes are important bio-molecules, which cleave the glycan backbone in the peptidoglycan polymer found in bacterial cell walls. The glycoside hydrolase (GH) family 22 C-type lysozyme, from the folivorous bird Opisthocomus hoazin (stinkbird), was expressed in Aspergillus oryzae, and a set of variants was produced. All variants were enzymatically active, including those designed to probe key differences between the Hoatzin enzyme and Hen Egg White lysozyme. Four variants showed improved thermostability at pH 4.7, compared to the wild type. The X-ray structure of the enzyme was determined in the apo form and in complex with chitin oligomers. Bioinformatic analysis of avian GH22 amino acid sequences showed that they separate out into three distinct subgroups (chicken-like birds, sea birds and other birds). The Hoatzin is found in the "other birds" group and we propose that this represents a new cluster of avian upper-gut enzymes.

The structural characterization of a prophage-encoded extracellular DNase from Streptococcus pyogenes.

The pathogenic bacterium Group A Streptococcus pyogenes produces several extracellular DNases that have been shown to facilitate invasive infection by evading the human host immune system. DNases degrade the chromatin in neutrophil extracellular traps, enabling the bacterium to evade neutrophil capture. Spd1 is a type I, nonspecific ßßα/metal-dependent nuclease from Streptococcus pyogenes, which is encoded by the SF370.1 prophage and is likely to be expressed as a result of prophage induction. We present here the X-ray structure of this DNase in the wild-type and Asn145Ala mutant form. Through structural and sequence alignments as well as mutagenesis studies, we have identified the key residues His121, Asn145 and Glu164, which are crucial for Spd1 nucleolytic activity and shown the active site constellation. Our wild-type structure alludes to the possibility of a catalytically blocked dimeric form of the protein. We have investigated the multimeric nature of Spd1 using size-exclusion chromatography with multi-angle light scattering (SEC-MALLS) in the presence and absence of the divalent metal ion Mg(2+), which suggests that Spd1 exists in a monomeric form in solution.

Rational design of a cyclohexanone dehydrogenase for enhanced α,ß-desaturation and substrate specificity.

The selective α,ß-desaturation of cyclic carbonyl compounds, which are found in the core of many steroid and bioactive molecules, using green chemistry is highly desirable. To achieve this task, we have for the first time described and solved the de novo structure of a member of the cyclohexanone dehydrogenase class of enzymes. The breadth of substrate specificity was investigated by assaying the cyclohexanone dehydrogenase, from Alicycliphilus denitrificans, against several cyclic ketones, lactones and lactams. To investigate substrate binding, a catalytic variant, Y195F, was generated and used to obtain a crystallographic complex with the natural substrate, cyclohexanone. This revealed substrate-active site interactions, as well as the proximity of the cofactor, flavin adenine dinucleotide, and enabled us to propose a mechanistic function to key amino acids. We then used molecular dynamic simulations to guide design to add functionality to the cyclohexanone dehydrogenase enzyme. The resulting W113A variant had overall improved enzyme activity and substrate scope, i.e., accepting the bulkier carbonyl compound, dihydrocoumarin. Structural analysis of the W113A variant revealed a broader, more open active site, which helped explain the modified substrate specificity. This work paves the way for future bespoke regioselective α,ß-desaturation in the synthesis of important bioactive molecules via rational enzyme engineering.

Structure and activity of the Streptococcus pyogenes family GH1 6-phospho-ß-glucosidase SPy1599.

The group A streptococcus Streptococcus pyogenes is the causative agent of a wide spectrum of invasive infections, including necrotizing fasciitis, scarlet fever and toxic shock syndrome. In the context of its carbohydrate chemistry, it is interesting that S. pyogenes (in this work strain M1 GAS SF370) displays a spectrum of oligosaccharide-processing enzymes that are located in close proximity on the genome but that the in vivo function of these proteins remains unknown. These proteins include different sugar transporters (SPy1593 and SPy1595), both GH125 α-1,6- and GH38 α-1,3-mannosidases (SPy1603 and SPy1604), a GH84 ß-hexosaminidase (SPy1600) and a putative GH2 ß-galactosidase (SPy1586), as well as SPy1599, a family GH1 `putative ß-glucosidase'. Here, the solution of the three-dimensional structure of SPy1599 in a number of crystal forms complicated by unusual crystallographic twinning is reported. The structure is a classical (ß/α)(8)-barrel, consistent with CAZy family GH1 and other members of the GH-A clan. SPy1599 has been annotated in sequence depositions as a ß-glucosidase (EC 3.2.1.21), but no such activity could be found; instead, three-dimensional structural overlaps with other enzymes of known function suggested that SPy1599 contains a phosphate-binding pocket in the active site and has possible 6-phospho-ß-glycosidase activity. Subsequent kinetic analysis indeed showed that SPy1599 has 6-phospho-ß-glucosidase (EC 3.2.1.86) activity. These data suggest that SPy1599 is involved in the intracellular degradation of 6-phosphoglycosides, which are likely to originate from import through one of the organism's many phosphoenolpyruvate phosphotransfer systems (PEP-PTSs).

The copper active site of CBM33 polysaccharide oxygenases.

The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme's three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61.

Edible flowers of Helichrysum italicum: Composition, nutritive value, and bioactivities.

Helichrysum italicum (H. italicum) is a halophyte shrub with bright yellow flowers with a strong curry-like aroma. The essential oils of H. italicum have been used in the production of cosmetics and pharmaceuticals, due to their antiallergic and anti-inflammatory properties. In the agri-food sector, H. italicum flowers can be used for seasoning and flavoring food, and as natural food preservatives. Here, we report on the composition, bioactive compounds, and nutritive value of H. italicum flowers. Flowers were mainly composed of carbohydrates (>80 % dry weight), followed by minerals (6.31 ± 0.95 % dw), protein (5.44 ± 0.35 % dw), and lipids (3.59 % ± 0.53 % dw). High percentages of Fe, Zn, Ca, and K were found in the flower material, along with a high content in antioxidants, polyphenols, and carotenoids, as corroborated by the nuclear magnetic resonance (NMR) data. Flowers were mainly composed of saturated fatty acids (SFAs) (54.50 ± 0.95 % of total FA), followed by polyunsaturated fatty acids (PUFAs) (37.73 ± 1.25 % of total FA) and monounsaturated fatty acids (MUFAs) (7.77 ± 0.34 %), as detected by gas chromatography mass spectrometry (GC-MS). The omega-6 PUFA linoleic acid (22.55 ± 0.76 % of total FA) was the most abundant fatty acid found. Flower extracts showed antimicrobial activity against Saccharomyces cerevisiae and Komagataella phaffii, as well as against Gram-negative (Klebsiella pneumoniae) and Gram-positive (Staphylococcus aureus) bacteria. H. italicum flower material was nontoxic to human intestinal Caco-2 model cells at concentrations up to 1.0 % w/v.

Crystal structures of a family 8 polysaccharide lyase reveal open and highly occluded substrate-binding cleft conformations.

Bacterial enzymatic degradation of glycosaminoglycans such as hyaluronan and chondroitin is facilitated by polysaccharide lyases. Family 8 polysaccharide lyase (PL8) enzymes contain at least two domains: one predominantly composed of α-helices, the α-domain, and another predominantly composed of ß-sheets, the ß-domain. Simulation flexibility analyses indicate that processive exolytic cleavage of hyaluronan, by PL8 hyaluronate lyases, is likely to involve an interdomain shift, resulting in the opening/closing of the substrate-binding cleft between the α- and ß-domains, facilitating substrate translocation. Here, the Streptomyces coelicolor A3(2) PL8 enzyme was recombinantly expressed in and purified from Escherichia coli and biochemically characterized as a hyaluronate lyase. By using X-ray crystallography its structure was solved in complex with hyaluronan and chondroitin disaccharides. These findings show key catalytic interactions made by the different substrates, and on comparison with all other PL8 structures reveals that the substrate-binding cleft of the S. coelicolor enzyme is highly occluded. A third structure of the enzyme, harboring a mutation of the catalytic tyrosine, created via site-directed mutagenesis, interestingly revealed an interdomain shift that resulted in the opening of the substrate-binding cleft. These results add further support to the proposed processive mechanism of action of PL8 hyaluronate lyases and may indicate that the mechanism of action is likely to be universally used by PL8 hyaluronate lyases.

Structure and mechanism of a bacterial beta-glucosaminidase having O-GlcNAcase activity.

O-GlcNAc is an abundant post-translational modification of serine and threonine residues of nucleocytoplasmic proteins. This modification, found only within higher eukaryotes, is a dynamic modification that is often reciprocal to phosphorylation. In a manner analogous to phosphatases, a glycoside hydrolase termed O-GlcNAcase cleaves O-GlcNAc from modified proteins. Enzymes with high sequence similarity to human O-GlcNAcase are also found in human pathogens and symbionts. We report the three-dimensional structure of O-GlcNAcase from the human gut symbiont Bacteroides thetaiotaomicron both in its native form and in complex with a mimic of the reaction intermediate. Mutagenesis and kinetics studies show that the bacterial enzyme, very similarly to its human counterpart, operates via an unusual 'substrate-assisted' catalytic mechanism, which will inform the rational design of enzyme inhibitors.

The structure of a family GH25 lysozyme from Aspergillus fumigatus.

Lysins are important biomolecules which cleave the bacterial cell-wall polymer peptidoglycan. They are finding increasing commercial and medical application. In order to gain an insight into the mechanism by which these enzymes operate, the X-ray structure of a CAZy family GH25 ;lysozyme' from Aspergillus fumigatus was determined. This is the first fungal structure from the family and reveals a modified alpha/beta-barrel-like fold in which an eight-stranded beta-barrel is flanked by three alpha-helices. The active site lies toward the bottom of a negatively charged pocket and its layout has much in common with other solved members of the GH25 and related GH families. A conserved active-site DXE motif may be implicated in catalysis, lending further weight to the argument that this glycoside hydrolase family operates via a ;substrate-assisted' catalytic mechanism.