Unlocking the hydrolytic mechanism of GH92 α-1,2-mannosidases: computation inspires using C-glycosides as Michaelis complex mimics

The conformational changes in a sugar moiety along the hydrolytic pathway are key to understand the mechanism of glycoside hydrolases (GHs) and to design new inhibitors. The two predominant itineraries for mannosidases go via OS2  B2,5  1S5 and 3S  3H4  1C4. For the CAZy family 92, the conformational itinerary was unknown. Published complexes of Bacteroides thetaiotaomicron GH92 catalyst with a S-glycoside and mannoimidazole indicate a 4C1  4H5/1S5  1S5 mechanism. However, as observed with the GH125 family, S-glycosides may not act always as good mimics of GH’s natural substrate. Here we present a cooperative study between computations and experiments where our results predict the E5  B2,5/1S5  1S5 pathway for GH92 enzymes. Furthermore, we demonstrate the Michaelis complex mimicry of a new kind of C-disaccharides, whose biochemical applicability was still a chimera.

RNase E endonuclease activity and its inhibition by pseudoridine

The conserved endoribonuclease RNase E dominates the dynamic landscape of RNA metabolism and underpins control mediated by small regulatory RNAs in diverse bacterial species. We explored the enzyme's hydrolytic mechanism, allosteric activation, and interplay with partner proteins in the multi-component RNA degradosome assembly. RNase E cleaves single-stranded RNA with preference to attack the phosphate located at the 5ʹ nucleotide preceding uracil, and we corroborate key interactions that select that base. Unexpectedly, RNase E activity is impeded strongly when the recognised uracil is isomerised to 5-ribosyluracil (pseudouridine), from which we infer the detailed geometry of the hydrolytic attack process. Kinetics analyses support models for recognition of secondary structure in substrates by RNase E and for allosteric auto-regulation. The catalytic power of the enzyme is boosted when it is assembled into the multi-enzyme RNA degradosome, most likely as a consequence of substrate channeling. Our results rationalize the origins of substrate preferences of RNase E and illuminate its catalytic mechanism, supporting the roles of allosteric domain closure and cooperation with other components of the RNA degradosome complex.

Structure of the E. coli agmatinase, SPEB

Agmatine amidinohydrolase, or agmatinase, catalyzes the conversion of agmatine to putrescine and urea. This enzyme is found broadly across kingdoms of life and plays a critical role in polyamine biosynthesis and the regulation of agmatine concentrations. Here we describe the high-resolution X-ray crystal structure of the E. coli agmatinase, SPEB. The data showed a relatively high degree of pseudomerohedral twinning, was ultimately indexed in the P31 space group and led to a final model with eighteen chains, corresponding to three full hexamers in the asymmetric unit. There was a solvent content of 38.5% and refined R/Rfree values of 0.166/0.216. The protein has the conserved fold characteristic of the agmatine ureohydrolase family and displayed a high degree of structural similarity among individual protomers. Two distinct peaks of electron density were observed in the active site of most of the eighteen chains of SPEB. As the activity of this protein is known to be dependent upon manganese and the fold is similar to other dinuclear metallohydrolases, these peaks were modeled as manganese ions. The orientation of the conserved active site residues, in particular those amino acids that participate in binding the metal ions and a pair of acidic residues (D153 and E274 in SPEB) that play a role in catalysis, are similar to other agmatinase and arginase enzymes and is consistent with a hydrolytic mechanism that proceeds via a metal-activated hydroxide ion.

Novel Cephalosporin Conjugates Display Potent and Selective Inhibition of IMP Type Metallo-β-Lactamases

In an attempt to exploit the hydrolytic mechanism by which β-lactamase enzymes degrade cephalosporins, we designed and synthesized a series of novel cephalosporin prodrugs aimed at delivering thiol-based inhibitors of metallo-β-lactamases (MBLs) in spatiotemporally controlled fashion. Notably, while enzyme-mediated hydrolysis of the β-lactam ring was found to occur, it was not accompanied by release of the thiol-based inhibitors. Nonetheless, the cephalosporin prodrugs, especially thiomandelic acid conjugate (<b>8</b>), demonstrated potent inhibition of IMP-type MBLs, with IC₅₀ values in the nanomolar range. In addition, conjugate <b>8</b> was also found to greatly reduce the MIC of meropenem against an IMP-28 producing clinical isolate of <i>K. pneumoniae</i>. The results of kinetic experiments indicate that these prodrugs inhibit IMP-type MBLs by acting as slowly turned-over substrates. Structure-activity relationship studies revealed that both phenyl and carboxyl moieties of <b>8</b> are crucial for its potency. Furthermore, modeling studies indicate that productive interactions of the thiomandelic acid moiety of <b>8</b> with residues Trp28 and Lys161 within the IMP active site may contribute to the observed inhibitory potency and selectivity.

Novel Cephalosporin Conjugates Display Potent and Selective Inhibition of IMP Type Metallo-β-Lactamases

In an attempt to exploit the hydrolytic mechanism by which β-lactamase enzymes degrade cephalosporins, we designed and synthesized a series of novel cephalosporin prodrugs aimed at delivering thiol-based inhibitors of metallo-β-lactamases (MBLs) in spatiotemporally controlled fashion. Notably, while enzyme-mediated hydrolysis of the β-lactam ring was found to occur, it was not accompanied by release of the thiol-based inhibitors. Nonetheless, the cephalosporin prodrugs, especially thiomandelic acid conjugate (<b>8</b>), demonstrated potent inhibition of IMP-type MBLs, with IC₅₀ values in the nanomolar range. In addition, conjugate <b>8</b> was also found to greatly reduce the MIC of meropenem against an IMP-28 producing clinical isolate of <i>K. pneumoniae</i>. The results of kinetic experiments indicate that these prodrugs inhibit IMP-type MBLs by acting as slowly turned-over substrates. Structure-activity relationship studies revealed that both phenyl and carboxyl moieties of <b>8</b> are crucial for its potency. Furthermore, modeling studies indicate that productive interactions of the thiomandelic acid moiety of <b>8</b> with residues Trp28 and Lys161 within the IMP active site may contribute to the observed inhibitory potency and selectivity.

Characterization and Therapeutic Potential of Bacteriophage-Encoded Polysaccharide Depolymerases with β Galactosidase Activity against Klebsiella pneumoniae K57 Capsular Type

Bacteriophages and phage enzymes are considered as possible alternatives to antibiotics in the treatment of infections caused by antibiotic-resistant bacteria. Due to the ability to cleave the capsular polysaccharides (CPS), one of the main virulence factors of Klebsiella pneumoniae, phage depolymerases, has potential in the treatment of K. pneumoniae infections. Here, we characterized in vivo two novel phage-encoded polysaccharide depolymerases as therapeutics against clinical isolates of K. pneumoniae. The depolymerases Dep_kpv79 and Dep_kpv767 encoded by Klebsiella phages KpV79 (Myoviridae; Jedunavirus) and KpV767 (Autographiviridae, Studiervirinae, Przondovirus), respectively, were identified as specific β-galactosidases that cleave the K. pneumoniae K57 type CPS by the hydrolytic mechanism. They were found to be highly effective at combating sepsis and hip infection caused by K. pneumoniae in lethal mouse models. Here, 80–100% of animals were protected against death by a single dose (e.g., 50 μg/mouse) of the enzyme injected 0.5 h after infection by K. pneumoniae strains of the K57 capsular type. The therapeutic effect of the depolymerases is because they strip the capsule and expose the underlying bacterium to the immune attack such as complement-mediated killing. These data provide one more confirmation that phage polysaccharide depolymerases represent a promising tool for antimicrobial therapy.

Structure of a GH51 α-L-arabinofuranosidase from Meripilus giganteus: conserved substrate recognition from bacteria to fungi

α-L-Arabinofuranosidases from glycoside hydrolase family 51 use a stereochemically retaining hydrolytic mechanism to liberate nonreducing terminal α-L-arabinofuranose residues from plant polysaccharides such as arabinoxylan and arabinan. To date, more than ten fungal GH51 α-L-arabinofuranosidases have been functionally characterized, yet no structure of a fungal GH51 enzyme has been solved. In contrast, seven bacterial GH51 enzyme structures, with low sequence similarity to the fungal GH51 enzymes, have been determined. Here, the crystallization and structural characterization of MgGH51, an industrially relevant GH51 α-L-arabinofuranosidase cloned from Meripilus giganteus, are reported. Three crystal forms were grown in different crystallization conditions. The unliganded structure was solved using sulfur SAD data collected from a single crystal using the I23 in vacuo diffraction beamline at Diamond Light Source. Crystal soaks with arabinose, 1,4-dideoxy-1,4-imino-L-arabinitol and two cyclophellitol-derived arabinose mimics reveal a conserved catalytic site and conformational itinerary between fungal and bacterial GH51 α-L-arabinofuranosidases.

Multi-omics analysis provides insights into lignocellulosic biomass degradation by Laetiporus sulphureus ATCC 52600

Background: Wood-decay basidiomycetes are effective for the degradation of highly lignified and recalcitrant substrates. Brown-rot strains produce carbohydrate-active enzymes involved in the degradation of lignocellulosic materials, along with a non-enzymatic mechanism via Fenton reaction. Differences in the lignocellulose metabolism among closely related brown rots are not completely understood. Here, a multi-omics approach provided a global understanding of the strategies employed by L. sulphureus ATCC 52600 in the degradation of lignocellulosic by-products derived from sugarcane and Eucalyptus. Results: To evidence the oxidative-hydrolytic mechanism, the Laetiporus sulphureus ATCC 52600 genome was sequenced and the response to lignocellulosic substrates was analyzed by transcriptomics and proteomics. The transcriptomic profile in response to a short cultivation period on in natura sugarcane bagasse revealed 128 out of 12,802 upregulated transcripts. The high upregulated transcripts included a set of redox enzymes along with hemicellulases. The exoproteome produced in response to extended time cultivation on Avicel, and steam-exploded sugarcane bagasse, sugarcane straw, and Eucalyptus (from Eucalyptus grandis) revealed 121 proteins. Contrasting with the mainly oxidative profile observed in the transcriptome, the secretomes showed a diverse hydrolytic repertoire including constitutive cellulases and hemicellulases, in addition to 19 upregulated proteins relative to glucose. The secretome produced on sugarcane bagasse was evaluated in the saccharification of pretreated sugarcane straw by supplementing a commercial cocktail. Additionally, growth analysis revealed that L. sulphureus ATCC 52600 has higher efficiency to assimilate glucose than other mono- and disaccharides.Conclusion: This study shows the singularity of L. sulphureus ATCC 52600 compared to other Polyporales brown rots, regarding the presence of cellobiohydrolase and peroxidase class II. The multi-omics analysis reinforces the oxidative-hydrolytic metabolism involved in lignocellulose deconstruction, providing insights into the overall mechanisms as well as specific proteins of each step.

Autographivirinae Bacteriophage Arno 160 Infects Pectobacterium carotovorum via Depolymerization of the Bacterial O-Polysaccharide

Phytopathogenic bacteria belonging to the Pectobacterium and Dickeya genera (soft-rot Pectobacteriaceae) are in the focus of agriculture-related microbiology because of their diversity, their substantial negative impact on the production of potatoes and vegetables, and the prospects of bacteriophage applications for disease control. Because of numerous amendments in the taxonomy of P. carotovorum, there are still a few studied sequenced strains among this species. The present work reports on the isolation and characterization of the phage infectious to the type strain of P. carotovorum. The phage Arno 160 is a lytic Podovirus representing a potential new genus of the subfamily Autographivirinae. It recognizes O-polysaccahride of the host strain and depolymerizes it in the process of infection using a rhamnosidase hydrolytic mechanism. Despite the narrow host range of this phage, it is suitable for phage control application.

The hydrolysis mechanism of a GH45 cellulase and its potential relation to lytic transglycosylase and expansin function

Family 45 glycoside hydrolases (GH45) are endoglucanases that are integral to cellulolytic secretomes, and their ability to break down cellulose has been successfully exploited in textile and detergent industries. In addition to their industrial relevance, understanding the molecular mechanism of GH45-catalyzed hydrolysis is of fundamental importance because of their structural similarity to cell wall–modifying enzymes such as bacterial lytic transglycosylases (LTs) and expansins present in bacteria, plants, and fungi. Our understanding of the catalytic itinerary of GH45s has been incomplete because a crystal structure with substrate spanning the −1 to +1 subsites is currently lacking. Here we constructed and validated a putative Michaelis complex in silico and used it to elucidate the hydrolytic mechanism in a GH45, Cel45A from the fungus Humicola insolens, via unbiased simulation approaches. These molecular simulations revealed that the solvent-exposed active-site architecture results in lack of coordination for the hydroxymethyl group of the substrate at the −1 subsite. This lack of coordination imparted mobility to the hydroxymethyl group and enabled a crucial hydrogen bond with the catalytic acid during and after the reaction. This suggests the possibility of a nonhydrolytic reaction mechanism when the catalytic base aspartic acid is missing, as is the case in some LTs (murein transglycosylase A) and expansins. We calculated reaction free energies and demonstrate the thermodynamic feasibility of the hydrolytic and nonhydrolytic reaction mechanisms. Our results provide molecular insights into the hydrolysis mechanism in HiCel45A, with possible implications for elucidating the elusive catalytic mechanism in LTs and expansins.