Hydrolase Family Research Articles

The role of the C domain in the thermostability of GH70 enzymes investigated by domain swapping

Abstract Sucrose-active enzymes belonging to the glycoside hydrolase (GH) family 70 are attractive tools for the synthesis of oligosaccharides, polysaccharides or glycoconjugates. However, their thermostability is an important issue for the development of robust and cost-effective enzyme-based processes. Indeed, GH70 enzymes are mesophilic and no thermophilic representatives have been described so far. Furthermore, structurally guided engineering is a challenge given the size of these proteins (120 to 250 kDa) and their organization in five domains. Herein, we have investigated the possible role of the domain C in the stability of GH70 enzymes. The alternansucrase (ASR) is the most stable enzyme of the GH70 family. Structural comparison of ASR to other GH70 enzymes highlighted the compactness of its domain C. We assumed that this atypical structure might be involved in the stability of this enzyme and decided to introduce this domain in another much less stable GH70 enzyme of known three-dimensional structure, the branching sucrase GBD-CD2. The chimeric GBD-CD2 exhibited a lower specific activity on sucrose substrate but its specificity was unchanged with the enzyme remaining specific for the branching of dextran via α-1,2 linkage formation. Interestingly, the chimera showed a higher melting temperature and residual activity than the wild-type enzyme after 10 min incubation at 30 °C showing that the domain C can affect GH70 enzyme stability and could be a potential target of both random or rational mutagenesis to further improve their stability.

Three-dimensional structure of xylonolactonase from Caulobacter crescentus: A mononuclear iron enzyme of the 6-bladed β-propeller hydrolase family.

Xylonolactonase Cc XylC from Caulobacter crescentus catalyzes the hydrolysis of the intramolecular ester bond of d‐xylonolactone. We have determined crystal structures of Cc XylC in complex with d‐xylonolactone isomer analogues d‐xylopyranose and (r)‐(+)‐4‐hydroxy‐2‐pyrrolidinone at high resolution. Cc XylC has a 6‐bladed β‐propeller architecture, which contains a central open channel having the active site at one end. According to our previous native mass spectrometry studies, Cc XylC is able to specifically bind Fe2+. The crystal structures, presented here, revealed an active site bound metal ion with an octahedral binding geometry. The side chains of three amino acid residues, Glu18, Asn146, and Asp196, which participate in binding of metal ion are located in the same plane. The solved complex structures allowed suggesting a reaction mechanism for intramolecular ester bond hydrolysis in which the major contribution for catalysis arises from the carbonyl oxygen coordination of the xylonolactone substrate to the Fe2+. The structure of Cc XylC was compared with eight other ester hydrolases of the β‐propeller hydrolase family. The previously published crystal structures of other β‐propeller hydrolases contain either Ca2+, Mg2+, or Zn2+ and show clear similarities in ligand and metal ion binding geometries to that of Cc XylC. It would be interesting to reinvestigate the metal binding specificity of these enzymes and clarify whether they are also able to use Fe2+ as a catalytic metal. This could further expand our understanding of utilization of Fe2+ not only in oxidative enzymes but also in hydrolases.

Structure of a bacterial α-1,2-glucosidase defines mechanisms of hydrolysis and substrate specificity in GH65 family hydrolases

Glycoside hydrolase family 65 (GH65) comprises glycoside hydrolases (GHs) and glycoside phosphorylases (GPs) that act on α-glucosidic linkages in oligosaccharides. All previously reported bacterial GH65 enzymes are GPs, whereas all eukaryotic GH65 enzymes known are GHs. In addition, to date, no crystal structure of a GH65 GH has yet been reported. In this study, we use biochemical experiments and X-ray crystallography to examine the function and structure of a GH65 enzyme from Flavobacterium johnsoniae (FjGH65A) that shows low amino acid sequence homology to reported GH65 enzymes. We found that FjGH65A does not exhibit phosphorolytic activity, but it does hydrolyze kojibiose (α-1,2-glucobiose) and oligosaccharides containing a kojibiosyl moiety without requiring inorganic phosphate. In addition, stereochemical analysis demonstrated that FjGH65A catalyzes this hydrolytic reaction via an anomer-inverting mechanism. The three-dimensional structures of FjGH65A in native form and in complex with glucose were determined at resolutions of 1.54 and 1.40 Å resolutions, respectively. The overall structure of FjGH65A resembled those of other GH65 GPs, and the general acid catalyst Glu472 was conserved. However, the amino acid sequence forming the phosphate-binding site typical of GH65 GPs was not conserved in FjGH65A. Moreover, FjGH65A had the general base catalyst Glu616 instead, which is required to activate a nucleophilic water molecule. These results indicate that FjGH65A is an α-1,2-glucosidase and is the first bacterial GH found in the GH65 family.

Polysaccharide utilization by a marine heterotrophic bacterium from the SAR92 clade.

SAR92 is one of the few examples of a widely distributed, abundant oligotroph that can be cultivated to study pathways of carbon oxidation in ocean systems. Genomic evidence for SAR92 suggests that this gammaproteobacterium might be a primary consumer of polysaccharides in the epipelagic zone, its main habitat. Here, we investigated cell growth, polysaccharide utilization gene expression, and carbohydrate-active enzyme abundance of a culturable SAR92 strain, HTCC2207, grown with different polysaccharides. Xylan and laminarin, two polysaccharides mainly produced by phytoplankton, supported the growth of HTCC2207 better than other polysaccharides. HTCC2207 possessed polysaccharide utilization loci (PULs) consisting of TonB-dependent receptor (TBDR) and glycoside hydrolase (GH) family genes. GH genes such as GH17 and GH3 presented no substrate-specificity and were induced by different sugar substrates, while expressions of GH16, GH10 and GH30 were enhanced in the glucose-treatment but suppressed in the polysaccharide-treatment, indicating complex polysaccharide utilization by HTCC2207. Metabolic pathways for laminarin and xylan were re-constructed in HTCC2207 based on the PULs genes and other predicted carbohydrate-active enzymes. This study reveals features of the epipelagic niche of SAR92 and provide insight into the biogeochemical cycling of labile, high-molecular carbohydrate compounds in the surface ocean.

Isolation, Identification and Genome Analysis of a Cellulose-Degrading Bacterium Bacillus aciditolerans YN-1T

Bacillus aciditolerans YN-1T was isolated from Jietou town, Yunnan Province, China and exhibited various lignocellulolytic activities which had the ability to decompose straw. In this work, we found that strain YN-1T was capable of degrading different types of straw and showed the highest efficiency to the degrading of corn straw. We also draw the draft genome sequence of strain YN-1T , which composed of 4,633,983 bp chromosome. Results showed that Bacillus aciditolerans YN-1T contains 3455 protein coding sequences and 249 were associated with the carbohydrate metabolism. Meanwhile, CAZymes analysis also showed that strain YN-1T contains 60 coding genes of glycoside hydrolase (GH) family, implying that Bacillus aciditolerans YN-1T has broad applications in the degradation of straw.

Mycobacterial Populations Partly Change the Proportions of the Cells Undergoing Asymmetric/Symmetric Divisions in Response to Glycerol Levels in Growth Medium.

Twenty to thirty percent of the septating mycobacterial cells of the mid-log phase population showed highly deviated asymmetric constriction during division (ACD), while the remaining underwent symmetric constriction during division (SCD). The ACD produced short-sized cells (SCs) and normal/long-sized cells (NCs) as the sister–daughter cells, but with significant differential susceptibility to antibiotic/oxidative/nitrite stress. Here we report that, at 0.2% glycerol, formulated in the Middlebrook 7H9 medium, a significantly high proportion of the cells were divided by SCD. When the glycerol concentration decreased to 0.1% due to cell-growth/division, the ACD proportion gradually increased until the ACD:SCD ratio reached ~50:50. With further decrease in the glycerol levels, the SCD proportion increased with concomitant decrease in the ACD proportion. Maintenance of glycerol at 0.1%, through replenishment, held the ACD:SCD proportion at ~50:50. Transfer of the cells from one culture with a specific glycerol level to the supernatant from another culture, with a different glycerol level, made the cells change the ACD:SCD proportion to that of the culture from which the supernatant was taken. RT-qPCR data showed the possibility of diadenosine tetraphosphate phosphorylase (MSMEG_2932), phosphatidylinositol synthase (MSMEG_2933), and a Nudix family hydrolase (MSMEG_2936) involved in the ACD:SCD proportion-change in response to glycerol levels. We also discussed its physiological significance.

Genomic analysis of a functional haloacid-degrading gene of Bacillus megaterium strain BHS1 isolated from Blue Lake (Mavi G\xf6l\xfc, Turkey)

PurposeBacillus megaterium strain BHS1, isolated from an alkaline water sample taken from Mavi Gölü (Blue Lake, Turkey), can grow on minimal medium containing 2,2-dichloropropionic acid. We characterized this bacterium at the genomic level.MethodsThe HiSeq platform was used to carry out genome sequencing, de novo assembly, and scaffolding with strain BHS1. Next, genome data were analyzed to demarcate DNA regions containing protein-coding genes and determine the function of certain BHS1 genes. Finally, results from a colorimetric chloride ion–release assay demonstrated that strain BHS1 produces dehalogenase.ResultsDe novo assembly of the BHS1 genomic sequence revealed a genome size of ~ 5.37 Mb with an average G+C content of 38%. The predicted nuclear genome harbors 5509 protein-coding genes, 1353 tRNA genes, 67 rRNA genes, and 6 non-coding (mRNA) genes. Genomic mapping of strain BHS1 revealed its amenability to synthesize two families of dehalogenases (Cof-type haloacid dehalogenase IIB family hydrolase and haloacid dehalogenase type II), suggesting that these enzymes can participate in the catabolism of halogenated organic acids. The mapping identified seven Na+/H+ antiporter subunits that are vital for adaptation of the bacterium to an alkaline environment. Apart from a pairwise analysis to the well-established L-2-haloacid dehalogenases, whole-cell analysis strongly suggested that the haloacid dehalogenase type II might act stereospecifically on L-2-chloropropionic acid, D,L-2-chloropropionic acid, and 2,2-dichloropropionic acid. Whole-cell studies confirmed the utilization of these three substrates and the gene’s role in dehalogenation.ConclusionsTo our knowledge, this is the first report of the full genome sequence for strain BHS1, which enabled the characterization of selected genes having specific metabolic activities and their roles in the biodegradation of halogenated compounds.

Isolation of cyanide-degrading bacteria and molecular characterization of its cyanide-degrading nitrilase.

Hydrogen cyanide is an industrially important chemical, and its annual production is more than 1.5 million tons. Because of its toxicity, the cyanide-containing effluents from industries have caused many environmental problems. Among various methods to treat the contaminated soils or water, the biological degradation is regarded to be promising. We isolated two cyanide-degrading microorganisms, Pedobacter sp. EBE-1 and Bacillus sp. EBE-2, from soil contaminated with cyanide. Among these bacteria, Bacillus sp. EBE-2 exhibited significantly a high cyanide-degrading ability. Bacillus sp. EBE-2 might be used for the remediation of cyanide contaminated water or soil. A nitrilase gene was cloned from Bacillus sp. EBE-2. Bacillus nitrilase was expressed in Escherichia coli and purified. Bacillus nitrilase exhibited cyanide-degrading activity as a large oligomer. Since formic acid formation from cyanide was observed, Bacillus nitrilase is likely to be a cyanide hydrolase. Although there exist various homologous enzymes annotated as carbon-nitrogen family hydrolases, this is the first report on the cyanide degrading activity. The structure and catalytic site of Bacillus nitrilase were studied by homology modeling and molecular docking simulation.

An efficient phthalate ester-degrading Bacillus subtilis: Degradation kinetics, metabolic pathway, and catalytic mechanism of the key enzyme.

Phthalate ester pollution in the environment and food chain is frequently reported. Microbial treatment is a green and efficient method for solving this problem. The isolation and systematic investigation of microorganisms generally recognized as safe (GRAS) will provide useful resources. A GRAS Bacillus subtilis strain, BJQ0005, was isolated from Baijiu fermentation starter and efficiently degraded phthalate esters (PAEs). The half-lives for di-isobutyl phthalate, di-butyl phthalate and di-(2-ethylhexyl) phthalate were 3.93, 4.28, and 25.49h, respectively, from the initial amount of 10mg per 10mL reaction mixture, which are records using wild-type strains. Genome sequencing and metabolic intermediate analysis generated the whole metabolic pathway. Eighteen enzymes from the α/β hydrolase family were expressed. Enzymes GTW28_09400 and GTW28_13725 were capable of single ester bond hydrolysis of PAEs, while GTW28_17760 hydrolyzed di-ester bonds of PAEs. Using molecular docking, a possible mechanism affecting enzymatic ester bond hydrolysis of mono-butyl phthalate was proposed of GTW28_17760. The carboxyl group generated by the first hydrolysis step interacted with histidine in the catalytic active center, which negatively affected enzymatic hydrolysis. Isolation and systematic investigation of the PAE degradation characteristics of B.subtilis will promote the green and safe treatment of PAEs in the environment and food industry.

Endolysins from Antarctic Pseudomonas Display Lysozyme Activity at Low Temperature.

Organisms specialized to thrive in cold environments (so-called psychrophiles) produce enzymes with the remarkable ability to catalyze chemical reactions at low temperature. Cold activity relies on adaptive changes in the proteins’ sequence and structural organization that result in high conformational flexibility. As a consequence of flexibility, several such enzymes are inherently heat sensitive. Cold-active enzymes are of interest for application in a number of bioprocesses, where cold activity coupled with easy thermal inactivation can be of advantage. We describe the biochemical and functional properties of two glycosyl hydrolases (named LYS177 and LYS188) of family 19 (GH19), identified in the genome of an Antarctic marine Pseudomonas. Molecular evolutionary analysis placed them in a group of characterized GH19 endolysins active on lysozyme substrates, such as peptidoglycan. Enzyme activity peaks at about 25–35 °C and 40% residual activity is retained at 5 °C. LYS177 and LYS188 are thermolabile, with Tm of 52 and 45 °C and half-lives of 48 and 12 h at 37 °C, respectively. Bioinformatics analyses suggest that low heat stability may be associated to temperature-driven increases in local flexibility occurring mainly in a specific region of the polypeptide that is predicted to contain hot spots for aggregation.

Discovery and development of a novel short-chain fatty acid ester synthetic biocatalyst under aqueous phase from Monascus purpureus isolated from Baijiu

Short-chain fatty acid esters are important flavor chemicals in Chinese traditional fermented Baijiu. Monascus purpureus was recognized as an important microorganism contributing to ester synthesis. However, the molecular basis for ester synthesis was still lacking. The present work combined genome sequencing, transcriptome sequencing, gene library construction, and enzyme engineering to discover a novel catalyst from M. purpureus (isolated from Baijiu fermentation starter). Enzyme LIP05, belonging to the α/β hydrolase family, was identified to synthesize short-chain fatty acid esters under aqueous phase. After deleting the lid domain of LIP05, the synthesis of ethyl pentanoate, ethyl hexanoate, ethyl octanoate, or ethyl decanoate was achieved. Ethyl octanoate with the highest conversion ratio of 93.7% was obtained with the assistance of ultrasound. The study reveals the molecular basis for synthesizing short-chain fatty acid esters by M. purpureus and will promote the application of the species or the enzyme in food industry.

A Novel Primer Mixture for GH48 Genes: Quantification and Identification of Truly Cellulolytic Bacteria in Biogas Fermenters.

Genomic studies revealed the glycoside hydrolases of family 48 (GH48) as a powerful marker for the identification of truly cellulolytic bacteria. Here we report an improved method for detecting cellulolytic bacteria in lab-scale biogas fermenters by using GH48 genes as a molecular marker in DNA and RNA samples. We developed a mixture of primers for the specific amplification of a GH48 gene region in a broad range of bacteria. Additionally, we built a manually curated reference database containing GH48 gene sequences directly linked to the corresponding taxonomic information. Phylogenetic correlation analysis of GH48 to 16S rRNA gene sequences revealed that GH48 gene sequences with 94% identity belong with high confidence to the same genus. Applying this analysis, GH48 amplicon reads revealed that at mesophilic fermenter conditions, 50–99% of the OTUs appear to belong to novel taxa. In contrast, at thermophilic conditions, GH48 gene sequences from the genus Hungateiclostridium dominated with 60–91% relative abundance. The novel primer combinations enabled detection and relative quantification of a wide spectrum of GH48 genes in cellulolytic microbial communities. Deep phylogenetic correlation analysis and a simplified taxonomic identification with the novel database facilitate identification of cellulolytic organisms, including the detection of novel taxa in biogas fermenters.

Identification of a Second Type of AHL-lactonase from Rhodococcus sp. BH4, belonging to the α/β Hydrolase Superfamily.

N-acyl-homoserine lactone (AHL)-mediated quorum sensing (QS) plays a major role in development of biofilms, which contribute to rise in infections and biofouling in water-related industries. Interference in QS, called quorum quenching (QQ), has recieved a lot of attention in recent years. Rhodococcus spp. are known to have prominent quorum quenching activity and in previous reports it was suggested that this genus possesses multiple QQ enzymes, but only one gene, qsdA, which encodes an AHL-lactonase belonging to phosphotriesterase family, has been identified. Therefore, we conducted a whole genome sequencing and analysis of Rhodococcus sp. BH4 isolated from a wastewater treatment plant. The sequencing revealed another gene encoding a QQ enzyme (named jydB) that exhibited a high AHL degrading activity. This QQ enzyme had a 46% amino acid sequence similarity with the AHL-lactonase (AidH) of Ochrobactrum sp. T63. HPLC analysis and AHL restoration experiments by acidification revealed that the jydB gene encodes an AHL-lactonase which shares the known characteristics of the α/β hydrolase family. Purified recombinant JydB demonstrated a high hydrolytic activity against various AHLs. Kinetic analysis of JydB revealed a high catalytic efficiency (kcat/KM) against C4-HSL and 3-oxo-C6 HSL, ranging from 1.88 × 106 to 1.45 × 106 M-1 s-1, with distinctly low KM values (0.16 - 0.24 mM). This study affirms that the AHL degrading activity and biofilm inhibition ability of Rhodococcus sp. BH4 may be due to the presence of multiple quorum quenching enzymes, including two types of AHL-lactonases, in addition to AHL-acylase and oxidoreductase, for which the genes have yet to be described.

Structural basis for the O-acetyltransferase function of the extracytoplasmic domain of OatA from Staphylococcus aureus

Many bacteria possess enzymes that modify the essential cell-wall polymer peptidoglycan by O-acetylation. This modification occurs in numerous Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, a common cause of human infections. O-Acetylation of peptidoglycan protects bacteria from the lytic activity of lysozyme, a mammalian innate immune enzyme, and as such is important for bacterial virulence. The O-acetylating enzyme in Gram-positive bacteria, O-acetyltransferase A (OatA), is a two-domain protein consisting of an N-terminal integral membrane domain and a C-terminal extracytoplasmic domain. Here, we present the X-ray crystal structure at 1.71 Å resolution and the biochemical characterization of the C-terminal domain of S. aureus OatA. The structure revealed that this OatA domain adopts an SGNH-hydrolase fold and possesses a canonical catalytic triad. Site-specific replacement of active-site amino acids revealed the presence of a water-coordinating aspartate residue that limits esterase activity. This residue, although conserved in staphyloccocal OatA and most other homologs, is not present in the previously characterized streptococcal OatA. These results provide insights into the mechanism of acetyl transfer in the SGNH/GDSL hydrolase family and highlight important evolutionary differences between homologous OatA enzymes. Furthermore, this study enhances our understanding of PG O-acetyltransferases, which could guide the development of novel antibacterial drugs to combat infections with multidrug-resistant bacterial pathogens.

Research Progress on bacterial cutinases for plastic pollution

Cutinases(3.1.1.74) are serine esterases that belong to the α/β hydrolase family. Such enzymes are usually produced by phytopathogenic microorganisms in order to penetrate their hosts. Cutinase can degrade the stratum corneum in the leaves or the keratin of the cork in the bark. Cutinase hydrolyzes soluble esters, insoluble triglycerides and various polyesters. In addition to the hydrolysis reaction, cutinase also shows synthetic activity and transester activity. Therefore, as a multifunctional enzyme, cutinase has many fields of application. In recent years, it has been found that cutinase can biodegrade plastic and biomoodifie synthetic fibers. Cutinase is the most important enzyme in solving plastic pollution.

The transcriptome of Pinus pinaster under Fusarium circinatum challenge

BackgroundFusarium circinatum, the causal agent of pitch canker disease, poses a serious threat to several Pinus species affecting plantations and nurseries. Although Pinus pinaster has shown moderate resistance to F. circinatum, the molecular mechanisms of defense in this host are still unknown. Phytohormones produced by the plant and by the pathogen are known to play a crucial role in determining the outcome of plant-pathogen interactions. Therefore, the aim of this study was to determine the role of phytohormones in F. circinatum virulence, that compromise host resistance.ResultsA high quality P. pinaster de novo transcriptome assembly was generated, represented by 24,375 sequences from which 17,593 were full length genes, and utilized to determine the expression profiles of both organisms during the infection process at 3, 5 and 10 days post-inoculation using a dual RNA-sequencing approach. The moderate resistance shown by Pinus pinaster at the early time points may be explained by the expression profiles pertaining to early recognition of the pathogen, the induction of pathogenesis-related proteins and the activation of complex phytohormone signaling pathways that involves crosstalk between salicylic acid, jasmonic acid, ethylene and possibly auxins. Moreover, the expression of F. circinatum genes related to hormone biosynthesis suggests manipulation of the host phytohormone balance to its own benefit.ConclusionsWe hypothesize three key steps of host manipulation: perturbing ethylene homeostasis by fungal expression of genes related to ethylene biosynthesis, blocking jasmonic acid signaling by coronatine insensitive 1 (COI1) suppression, and preventing salicylic acid biosynthesis from the chorismate pathway by the synthesis of isochorismatase family hydrolase (ICSH) genes. These results warrant further testing in F. circinatum mutants to confirm the mechanism behind perturbing host phytohormone homeostasis.

Genome-wide annotation, comparison and functional genomics of carbohydrate-active enzymes in legumes infecting Fusarium oxysporum formae speciales

ABSTRACTFusarium wilt caused by soil borne ascomycetes fungi Fusarium oxysporum which has host-specific forms known as formae speciales (ff. spp.), apparently requires plant cell wall degrading enzymes (PCWDE) for successful invasion. In this study, 12 F. oxysporum ff. spp. were taken for genome-wide annotation and comparative analysis of CAZymes, with an assessment of secretory PCWDE and orthologues identification in the three legumes infecting ff. spp. Further, transcriptomic analysis in two legumes infecting ff. spp. using publically available data was also done. The comparative studies showed Glycoside hydrolase (GH) families to be abundant and Principle Component Analysis (PCA) formed two distinct clusters of ff. spp. based on the CAZymes modules and families. Nearly half of the CAZymes in the legumes infecting ff. spp. coded for signal peptides. The orthologue clusters of secretory CAZymes common in all the three legume infecting ff. spp. mostly belonged to families of AA9, GH28, CE5 and PL1 and the expression analysis revealed the abundant PCWDE were differentially expressed in these legumes infecting ff. spp. Therefore, this study gave an insight into the distribution of CAZymes especially extracellular PCWDE in legumes infecting ff. spp. with further shedding light onto some of the key PCWDE families through differential expression analysis.

Algorithm-based coevolution network identification reveals key functional residues of the α/β hydrolase subfamilies.

Covariant residues identified by computational algorithms have provided new insights into enzyme evolutionary routes. However, the reliability and accuracy of routine statistical coupling analysis (SCA) are unable to satisfy the needs of protein engineering because SCA depends only on sequence information. Here, we set up a new SCA algorithm, SCA.SIM, by integrating structure information and MD simulation data. The more reliable covariant residues with high-quality scores are obtained from sequence alignment weighted by residual movement for eight related subfamilies, belonging to α/β hydrolase family, with Candida antarctica lipase B (CALB). The 38 predicted covariant residues are tested for function by high-throughput quantitative evaluation in combination with activity and thermostability assays of a mutant library and deep sequencing. Based on the landscapes of both activity and thermostability, most mutants play key roles in catalysis, and some mutants gain 2.4- to 6-fold increase in half-life at 50°C and 9- to 12-fold improvement in catalytic efficiency. The activity of double mutants for A225F/T103A is higher than those of A225F and T103A which means that SCA.SIM method might be useful for identifying the allosteric coupling. The SCA.SIM algorithm can be used for protein coevolution and enzyme engineering research.

Functional annotation of serine hydrolases in the asexual erythrocytic stage of Plasmodium falciparum

Enzymes of the serine hydrolase superfamily are ubiquitous, highly versatile catalysts that mediate a wide variety of metabolic reactions in eukaryotic cells, while also being amenable to selective inhibition. We have employed a fluorophosphonate-based affinity capture probe and mass spectrometry to explore the expression profile and metabolic roles of the 56-member P. falciparum serine hydrolase superfamily in the asexual erythrocytic stage of P. falciparum. This approach provided a detailed census of active serine hydrolases in the asexual parasite, with identification of 21 active serine hydrolases from α/β hydrolase, patatin, and rhomboid protease families. To gain insight into their functional roles and substrates, the pan-lipase inhibitor isopropyl dodecylfluorophosphonate was employed for competitive activity-based protein profiling, leading to the identification of seven serine hydrolases with potential lipolytic activity. We demonstrated how a chemoproteomic approach can provide clues to the specificity of serine hydrolases by using a panel of neutral lipase inhibitors to identify an enzyme that reacts potently with a covalent monoacylglycerol lipase inhibitor. In combination with existing phenotypic data, our studies define a set of serine hydrolases that likely mediate critical metabolic reactions in asexual parasites and enable rational prioritization of future functional characterization and inhibitor development efforts.

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.