Multisubstrate specificity shaped the complex evolution of the aminotransferase family across the tree of life.

Aminotransferases (ATs) are an ancient enzyme family that play central roles in core nitrogen metabolism, essential to all organisms. However, many of the AT enzyme functions remain poorly defined, limiting our fundamental understanding of the nitrogen metabolic networks that exist in different organisms. Here, we traced the deep evolutionary history of the AT family by analyzing AT enzymes from 90 species spanning the tree of life (ToL). We found that each organism has maintained a relatively small and constant number of ATs. Mapping the distribution of ATs across the ToL uncovered that many essential AT reactions are carried out by taxon-specific AT enzymes due to wide-spread nonorthologous gene displacements. This complex evolutionary history explains the difficulty of homology-based AT functional prediction. Biochemical characterization of diverse aromatic ATs further revealed their broad substrate specificity, unlike other core metabolic enzymes that evolved to catalyze specific reactions today. Interestingly, however, we found that these AT enzymes that diverged over billion years share common signatures of multisubstrate specificity by employing different nonconserved active site residues. These findings illustrate that AT family enzymes had leveraged their inherent substrate promiscuity to maintain a small yet distinct set of multifunctional AT enzymes in different taxa. This evolutionary history of versatile ATs likely contributed to the establishment of robust and diverse nitrogen metabolic networks that exist throughout the ToL. The study provides a critical foundation to systematically determine diverse AT functions and underlying nitrogen metabolic networks across the ToL.

Orthology-based analysis helps map evolutionary diversification and predict substrate class use of BAHD acyltransferases.

Large enzyme families catalyze metabolic diversification by virtue of their ability to use diverse chemical scaffolds. How enzyme families attain such functional diversity is not clear. Furthermore, duplication and promiscuity in such enzyme families limits their functional prediction, which has produced a burgeoning set of incompletely annotated genes in plant genomes. Here, we address these challenges using BAHD acyltransferases as a model. This fast-evolving family expanded drastically in land plants, increasing from one to five copies in algae to approximately 100 copies in diploid angiosperm genomes. Compilation of >160 published activities helped visualize the chemical space occupied by this family and define eight different classes based on structural similarities between acceptor substrates. Using orthologous groups (OGs) across 52 sequenced plant genomes, we developed a method to predict BAHD acceptor substrate class utilization as well as origins of individual BAHD OGs in plant evolution. This method was validated using six novel and 28 previously characterized enzymes and helped improve putative substrate class predictions for BAHDs in the tomato genome. Our results also revealed that while cuticular wax and lignin biosynthetic activities were more ancient, anthocyanin acylation activity was fixed in BAHDs later near the origin of angiosperms. The OG-based analysis enabled identification of signature motifs in anthocyanin-acylating BAHDs, whose importance was validated via molecular dynamic simulations, site-directed mutagenesis and kinetic assays. Our results not only describe how BAHDs contributed to evolution of multiple chemical phenotypes in the plant world but also propose a biocuration-enabled approach for improved functional annotation of plant enzyme families.

Characterization and Expression Analyses of Callose Synthase Enzyme (Cals) Family Genes in Maize (Zea mays L.).

The callose synthase enzyme genes (Cals) generally plays an important role in resisting to environmental stresses as well as in regulating the microspore development of higher plant. However till now, few researches about ZmCals genes have been reported in maize. In this study, ten ZmCals genes were identified, and they are distributed on four chromosomes in maize. All ZmCals proteins contain Glucan-synthase-domain and Fks1-domain. RNA-seq data from public databases were analyzed and the result suggested that ZmCals involved in the development of various tissues, and a strong expression presented especially in young tissue. qRT-PCR analysis shown that most of ZmCals are highly expressed in root, stem and leaf at jointing stage (V6 stage) with maize inbred line B73. Seven out of 10 ZmCals genes display higher expression during maize anther development especially from stage 6 to stage 8b, the dynamic accumulation process of callose is also observed during these period with aniline blue staining. Above results indicated multiple ZmCals may participate in the deposition of callose in maize anther. Therefore, ZmCals are necessary not only for reproductive organ but also for nutritive organ during maize growth and development. This study lays certain foundation for further investigating the roles of the callose synthase enzymes genes in maize.

Alteration of the Catalytic Reaction Trajectory of a Vicinal Oxygen Chelate Enzyme by Directed Evolution.

The vicinal oxygen chelate (VOC) metalloenzyme superfamily catalyzes a highly diverse set of reactions with the mechanism characterized by the bidentate coordination of vicinal oxygen atoms to metal ion centers, but there remains a lack of a platform to steer the reaction trajectories, especially for o-quinone metabolizing pathways. Herein, we present the directed-evolution-enabled bifunctional turnover of ChaP, which is a homotetramer and represents an unprecedented VOC enzyme class. Unlike the ChaP catalysis of extradiol-like o-quinone cleavage and concomitant α-keto acid decarboxylation, a group of ChaP variants (CVs) catalyze intradiol-like o-quinone deconstruction and CO2 liberation from the resulting o-hydroxybenzoic acid scaffolds with high regioselectivity. Enzyme crystal structures, labeling experiments and computational simulations corroborated that the D49L mutation allows the metal ion to change its coordination with the tyrosine phenoxy atoms in different monomers, thereby altering the reaction trajectory with the regiospecificity further improved by the follow-up replacement of the Y92 residue with any of alanine, glycine, threonine, and serine. The study highlights the unpredicted catalytic versatility and enzymatic plasticity of VOC enzymes with biotechnological significance.

Novel insights into cytochrome P450 enzyme and solute carrier families in cadmium-induced liver injury of pigs.

Cadmium (Cd) is a typical pollutant and carcinogen in environment. Exposure assessment of contaminants is an important component of occupational and environmental epidemiological studies. Early studies of Cd have focused on aquatic animals, chickens and rats. However, toxicological evaluation of Cd in pigs has not been reported. Therefore, twelve pigs were randomly divided into two groups (n = 6): the control group and the Cd group (Cd content: 15 ± 0.242 mg/kg feed) in this study, the experimental period was 30 d, and the toxic effects of Cd on the liver of weanling piglets were examined by antioxidant function, liver function, Cd content, histological examination and transcriptomics. The results showed that the changes of antioxidant function, liver function and Cd content were significant in the liver. Transcriptional profiling results showed that 399 differentially expressed genes (DEGs) were significantly up-regulated while 369 DEGs were remarkably down-regulated in Cd group, and which were concentrated in three ontologies: molecular function, cellular component and biological processes. Interestingly, significant changes in some genes of the cytochrome P450 enzyme (CYP450) and solute carrier (SLC) families have been observed and were consistent with qRT-PCR results. In conclusion, Cd could cause liver injury in weanling piglets and change the transcriptomic characteristics of liver. CYP450 and SLC families play an indispensable role in Cd-mediated hepatotoxicity. Importantly, changes in mRNA levels of CYP2B22, CYP7A1, CYP8B1, SLC26A8, SLC11A1, SLC27A2 and SLC22A7 induced by Cd have been reported for the first time. Our findings will provide a new insight for better assessing the mechanism of Cd toxicity to the liver.

An α/ß-Hydrolase Fold Subfamily Comprising Pseudomonas Quinolone Signal-Cleaving Dioxygenases.

The quinolone ring is a common core structure of natural products exhibiting antimicrobial, cytotoxic, and signaling activities. A prominent example is the Pseudomonas quinolone signal (PQS), a quorum-sensing signal molecule involved in the regulation of virulence of Pseudomonas aeruginosa The key reaction to quinolone inactivation and biodegradation is the cleavage of the 3-hydroxy-4(1H)-quinolone ring, catalyzed by dioxygenases (HQDs), which are members of the α/ß-hydrolase fold superfamily. The α/ß-hydrolase fold core domain consists of a ß-sheet surrounded by α-helices, with an active site usually containing a catalytic triad comprising a nucleophilic residue, an acidic residue, and a histidine. The nucleophile is located at the tip of a sharp turn, called the "nucleophilic elbow." In this work, we developed a search workflow for the identification of HQD proteins from databases. Search and validation criteria include an [H-x(2)-W] motif at the nucleophilic elbow, an [HFP-x(4)-P] motif comprising the catalytic histidine, the presence of a helical cap domain, the positioning of the triad's acidic residue at the end of ß-strand 6, and a set of conserved hydrophobic residues contributing to the substrate cavity. The 161 candidate proteins identified from the UniProtKB database originate from environmental and plant-associated microorganisms from all domains of life. Verification and characterization of HQD activity of 9 new candidate proteins confirmed the reliability of the search strategy and suggested residues correlating with distinct substrate preferences. Among the new HQDs, PQS dioxygenases from Nocardia farcinica, N. cyriacigeorgica, and Streptomyces bingchenggensis likely are part of a catabolic pathway for alkylquinolone utilization.IMPORTANCE Functional annotation of protein sequences is a major requirement for the investigation of metabolic pathways and the identification of sought-after biocatalysts. To identify heterocyclic ring-cleaving dioxygenases within the huge superfamily of α/ß-hydrolase fold proteins, we defined search and validation criteria for the primarily motif-based identification of 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQD). HQDs are key enzymes for the inactivation of metabolites, which can have signaling, antimicrobial, or cytotoxic functions. The HQD candidates detected in this study occur particularly in environmental and plant-associated microorganisms. Because HQDs active toward the Pseudomonas quinolone signal (PQS) likely contribute to interactions within microbial communities and modulate the virulence of Pseudomonas aeruginosa, we analyzed the catalytic properties of a PQS-cleaving subset of HQDs and specified characteristics to identify PQS-cleaving dioxygenases within the HQD family.

Draft genome of Streptomyces sp. strain 130 and functional analysis of extracellular enzyme producing genes.

Streptomyces sp. strain 130 possesses multiple uncharacterized extracellular enzyme producing genes. Enzymes from these genes may fulfil the intense demand of stable and effective extracellular enzymes in various industries. Taxonomy of Streptomyces sp. strain 130 was validated by FAME analysis. Strain 130 was screened for the presence of chitinase producing genes of family 18 and 19 using SC1F/SC2R and F19F2/F19R primer sets respectively. Whole genome sequencing was done using Illumina Next Seq 500 system. In the analysis of draft genome of Streptomyces sp. strain 130, the genome size was found to be 7.1 Mb. Blastn and NCBI-conserved domain search tool were used to find similarity percentage with genes in existing database and enzyme family respectively. Ten chitinase, six xylanase and one cellulase producing genes were present in draft genome. Among the ten chitinase producing genes, two were belonging to GH19 family and other eight to GH18 family chitinase. Six out of ten chitinase producing genes were uncharacterized and one belonged to family GH18_PF-ChiA (PF-ChiA is a chitinase found in the hyperthermophilic archaea, prokaryotes). In case of xylanase, four out of six (GH9, 43, 10 and 11 enzyme family) were not showing nucleotide based similarity with any characterized gene. The study of reported genome sequence will help us to identify gene sequence of characterized and uncharacterized extracellular enzyme producing genes. Cloning of each gene and enzyme activity assay of their products will reveal the activity and stability at different variables; and resulting products may have huge applications at industrial scale.

The actinobacterium Tsukamurella paurometabola has a functionally divergent arylamine N-acetyltransferase (NAT) homolog.

Actinobacteria in the Tsukamurella genus are aerobic, high-GC, Gram-positive mycolata, considered as opportunistic pathogens and isolated from various environmental sources, including sites contaminated with oil, urban or industrial waste and pesticides. Although studies look into xenobiotic biotransformation by Tsukamurella isolates, the relevant enzymes remain uncharacterized. We investigated the arylamine N-acetyltransferase (NAT) enzyme family, known for its role in the xenobiotic metabolism of prokaryotes and eukaryotes. Xenobiotic sensitivity of Tsukamurella paurometabola type strain DSM 20162T was assessed, followed by cloning, recombinant expression and functional characterization of its single NAT homolog (TSUPD)NAT1. The bacterium appeared quite robust against chloroanilines, but more sensitive to 4-anisidine and 2-aminophenol. However, metabolic activity was not evident towards those compounds, presumably due to mechanisms protecting cells from xenobiotic entry. Of the pharmaceutical arylhydrazines tested, hydralazine was toxic, but the bacterium was less sensitive to isoniazid, a drug targeting mycolic acid biosynthesis in mycobacteria. Although (TSUPD)NAT1 protein has an atypical Cys-His-Glu (instead of the expected Cys-His-Asp) catalytic triad, it is enzymatically active, suggesting that this deviation is likely due to evolutionary adaptation potentially serving a different function. The protein was indeed found to use malonyl-CoA, instead of the archetypal acetyl-CoA, as its preferred donor substrate. Malonyl-CoA is important for microbial biosynthesis of fatty acids (including mycolic acids) and polyketide chains, and the corresponding enzymatic systems have common evolutionary histories, also linked to xenobiotic metabolism. This study adds to accummulating evidence suggesting broad phylogenetic and functional divergence of microbial NAT enzymes that goes beyond xenobiotic metabolism and merits investigation.

Reductive activation of E. coli respiratory nitrate reductase.

Over the past decades, a number of authors have reported the presence of inactive species in as-prepared samples of members of the Mo/W-bisPGD enzyme family. This greatly complicated the spectroscopic studies of these enzymes, since it is impossible to discriminate between active and inactive species on the basis of the spectroscopic signatures alone. Escherichia coli nitrate reductase A (NarGHI) is a member of the Mo/W-bisPGD family that allows anaerobic respiration using nitrate as terminal electron acceptor. Here, using protein film voltammetry on NarGH films, we show that the enzyme is purified in a functionally heterogeneous form that contains between 20 and 40% of inactive species that activate the first time they are reduced. This activation proceeds in two steps: a non-redox reversible reaction followed by an irreversible reduction. By carefully correlating electrochemical and EPR spectroscopic data, we show that neither the two major Mo(V) signals nor those of the two FeS clusters that are the closest to the Mo center are associated with the two inactive species. We also conclusively exclude the possibility that the major "low-pH" and "high-pH" Mo(V) EPR signatures correspond to species in acid-base equilibrium.

The NADH:flavin oxidoreductase Nox from Rhodococcus erythropolis MI2 is the key enzyme of 4,4'-dithiodibutyric acid degradation.

The reduction of the disulphide bond is the initial catabolic step of the microbial degradation of the organic disulphide 4,4'-dithiodibutyric acid (DTDB). Previously, an NADH:flavin oxidoreductase from Rhodococcus erythropolis MI2 designated as NoxMI2 , which belongs to the old yellow enzyme (OYE) family, was identified. In the present study, it was proven that NoxMI2 has the ability to cleave the sulphur-sulphur bond in DTDB. In silico analysis revealed high sequence similarities to proteins of the flavin mononucleotide (FMN) reductase family identified in many strains of R. erythropolis. Therefore, nox was heterologously expressed in the pET23a(+) expression system using Escherichia coli strain BL21(DE3) pLysS, which effectively produces soluble active NoxMI2 . NoxMI2 showed a maximum specific activity (Vmax ) of 3·36 µmol min-1  mg-1 corresponding to a kcat of 2·5 s-1 and an apparent substrate Km of 0·6 mmol l-1 , when different DTDB concentrations were applied. No metal cofactors were required. Moreover, NoxMI2 had very low activity with other sulphur-containing compounds like 3,3'-dithiodipropionic acid (8·0%), 3,3'-thiodipropionic acid (7·6%) and 5,5'-dithiobis(2-nitrobenzoic acid) (8·0%). The UV/VIS spectrum of NoxMI2 revealed the presence of the cofactor FMN. Based on results obtained, NoxMI2 adds a new physiological substrate and mode of action to OYE members. SIGNIFICANCE AND IMPACT OF THE STUDY: It was unequivocally demonstrated in this study that an NADH:flavin oxidoreductase from Rhodococcus erythropolis MI2 (NoxMI2 ) is able to cleave the xenobiotic disulphide 4,4'-dithiodibutyric acid (DTDB) into two molecules of 4-mercaptobutyric acid (4MB) with concomitant consumption of NADH. NoxMI2 showed a high substrate specificity as well as high heat stability. This study provides the first detailed characterization of the initial cleavage of DTDB, which is considered as a promising polythioester precursor.

Crystal structure analysis of a bacterial aryl acylamidase belonging to the amidase signature enzyme family.

The atomic structure of a bacterial aryl acylamidase (EC 3.5.1.13; AAA) is reported and structural features are investigated to better understand the catalytic profile of this enzyme. Structures of AAA were determined in its native form and in complex with the analgesic acetanilide, p-acetaminophenol, at 1.70 Å and 1.73 Å resolutions, respectively. The overall structural fold of AAA was identified as an α/ß fold class, exhibiting an open twisted ß-sheet core surrounded by α-helices. The asymmetric unit contains one AAA molecule and the monomeric form is functionally active. The core structure enclosing the signature sequence region, including the canonical Ser-cisSer-Lys catalytic triad, is conserved in all members of the Amidase Signature enzyme family. The structure of AAA in a complex with its ligand reveals a unique organization in the substrate-binding pocket. The binding pocket consists of two loops (loop1 and loop2) in the amidase signature sequence and one helix (α10) in the non-amidase signature sequence. We identified two residues (Tyr(136) and Thr(330)) that interact with the ligand via water molecules, and a hydrogen-bonding network that explains the catalytic affinity over various aryl acyl compounds. The optimum activity of AAA at pH > 10 suggests that the reaction mechanism employs Lys(84) as the catalytic base to polarize the Ser(187) nucleophile in the catalytic triad.

The shikimate dehydrogenase family: functional diversity within a conserved structural and mechanistic framework.

Shikimate dehydrogenase (SDH) catalyzes the NADPH-dependent reduction of 3-deydroshikimate to shikimate, an essential reaction in the biosynthesis of the aromatic amino acids and a large number of other secondary metabolites in plants and microbes. The indispensible nature of this enzyme makes it a potential target for herbicides and antimicrobials. SDH is the archetypal member of a large protein family, which contains at least four additional functional classes with diverse metabolic roles. The different members of the SDH family share a highly similar three-dimensional structure and utilize a conserved catalytic mechanism, but exhibit distinct substrate preferences, making the family a particularly interesting system for studying modes of substrate recognition used by enzymes. Here, we review our current understanding of the biochemical and structural properties of each of the five previously identified SDH family functional classes.

Structure of choline oxidase in complex with the reaction product glycine betaine.

Choline oxidase from Arthrobacter globiformis, which is involved in the biosynthesis of glycine betaine from choline, has been extensively characterized in its mechanistic and structural properties. Despite the knowledge gained on the enzyme, the details of substrate access to the active site are not fully understood. The `loop-and-lid' mechanism described for the glucose-methanol-choline enzyme superfamily has not been confirmed for choline oxidase. Instead, a hydrophobic cluster on the solvent-accessible surface of the enzyme has been proposed by molecular dynamics to control substrate access to the active site. Here, the crystal structure of the enzyme was solved in complex with glycine betaine at pH 6.0 at 1.95 Šresolution, allowing a structural description of the ligand-enzyme interactions in the active site. This structure is the first of choline oxidase in complex with a physiologically relevant ligand. The protein structures with and without ligand are virtually identical, with the exception of a loop at the dimer interface, which assumes two distinct conformations. The different conformations of loop 250-255 define different accessibilities of the proposed active-site entrance delimited by the hydrophobic cluster on the other subunit of the dimer, suggesting a role in regulating substrate access to the active site.

Loss of the two major leaf isoforms of sucrose-phosphate synthase in Arabidopsis thaliana limits sucrose synthesis and nocturnal starch degradation but does not alter carbon partitioning during photosynthesis.

Sucrose (Suc)-phosphate synthase (SPS) catalyses one of the rate-limiting steps in the synthesis of Suc in plants. The Arabidopsis genome contains four annotated SPS genes which can be grouped into three different families (SPSA1, SPSA2, SPSB, and SPSC). However, the functional significance of this multiplicity of SPS genes is as yet only poorly understood. All four SPS isoforms show enzymatic activity when expressed in yeast although there is variation in sensitivity towards allosteric effectors. Promoter-reporter gene analyses and quantitative real-time reverse transcription-PCR studies indicate that no two SPS genes have the same expression pattern and that AtSPSA1 and AtSPSC represent the major isoforms expressed in leaves. An spsa1 knock-out mutant showed a 44% decrease in leaf SPS activity and a slight increase in leaf starch content at the end of the light period as well as at the end of the dark period. The spsc null mutant displayed reduced Suc contents towards the end of the photoperiod and a concomitant 25% reduction in SPS activity. In contrast, an spsa1/spsc double mutant was strongly impaired in growth and accumulated high levels of starch. This increase in starch was probably not due to an increased partitioning of carbon into starch, but was rather caused by an impaired starch mobilization during the night. Suc export from excised petioles harvested from spsa1/spsc double mutant plants was significantly reduced under illumination as well as during the dark period. It is concluded that loss of the two major SPS isoforms in leaves limits Suc synthesis without grossly changing carbon partitioning in favour of starch during the light period but limits starch degradation during the dark period.

Investigation of amino acids related to Staphylococcus saprophyticus AG1 EstAG1 carboxylesterase catalytic function revealed a new family of bacterial lipolytic enzymes.

Most of the lipolytic enzymes (carboxylesterases, EC 3.1.1.1 and triacylglycerol acylhydrolases, EC 3.1.1.3) originate from bacteria and form a large group of functionally important enzymes that are also well known for their use in multiple biotechnology sectors. Rapid and increasing amount of bacterial lipolytic enzymes being discovered and characterized led to a necessity to classify them. More than twenty years ago bacterial lipolytic enzymes were originally classified into eight families and six true lipase sub-families based on the differences in their amino acid sequences and biochemical properties. Later, this classification was comprehensively updated to 19 families with eight subfamilies, and more recently, employing deeper comparative analysis methods, classification expanded to 35 families and 11 subfamilies. Bacterial lipolytic enzymes that cannot be classified into currently existing families are still being discovered. This work provides site-directed mutagenesis and differential scanning fluorimetry based investigation of catalytic function-related amino acids of previously discovered and characterized EstAG1 carboxylesterase from Staphylococcus saprophyticus AG1. Experimental results obtained in this work revealed that EstAG1 carboxylesterase can be placed into a new family of bacterial lipolytic enzymes.

Thioesterase enzyme families: Functions, structures, and mechanisms.

Thioesterases are enzymes that hydrolyze thioester bonds in numerous biochemical pathways, for example in fatty acid synthesis. This work reports known functions, structures, and mechanisms of updated thioesterase enzyme families, which are classified into 35 families based on sequence similarity. Each thioesterase family is based on at least one experimentally characterized enzyme, and most families have enzymes that have been crystallized and their tertiary structure resolved. Classifying thioesterases into families allows to predict tertiary structures and infer catalytic residues and mechanisms of all sequences in a family, which is particularly useful because the majority of known protein sequence have no experimental characterization. Phylogenetic analysis of experimentally characterized thioesterases that have structures with the two main structural folds reveal convergent and divergent evolution. Based on tertiary structure superimposition, catalytic residues are predicted.

Genome-wide analysis of the callose enzyme families of fertile and sterile flower buds of the Chinese cabbage (Brassica rapa L. ssp. pekinensis).

Callose is a ß-1,3-glucan commonly found in higher plants that plays an important role in regulating plant pollen development. It is synthesized by glucan synthase-like (GSL) and is degraded by the enzyme endo-1,3-ß-glucosidase. However, genome-wide analyses of callose GSL and endo-1,3-ß-glucosidase enzymes in fertile and sterile flower buds of Chinese cabbage have not yet been reported. Here, we show that delayed callose degradation at the tetrad stage may be the main cause of microspore abortion in Chinese cabbage with nuclear sterility near-isogenic line '10L03'. Fifteen callose GSLs and 77 endo-1,3-ß-glucosidase enzymes were identified in Chinese cabbage. Phylogenetic, gene structural and chromosomal analyses revealed that the expansion occurred due to three polyploidization events of these two gene families. Expression pattern analysis showed that the GSL and endo-1,3-ß-glucosidase enzymes are involved in the development of various tissues and that the genes functionally diverged during long-term evolution. Relative gene expression analysis of Chinese cabbage flowers at different developmental stages showed that high expression of the synthetic enzyme BraA01g041620 and low expression of AtA6-homologous genes (BraA04g008040, BraA07g009320, BraA01g030220 and BraA03g040850) and two other genes (BraA10g020080 and BraA05g038340) for degrading enzymes in the meiosis and tetrad stages may cause nuclear sterility in the near-isogenic line '10L03'. Overall, our data provide an important foundation for comprehending the potential roles of the callose GSL and endo-1,3-ß-glucosidase enzymes in regulating pollen development in Chinese cabbage.

Expression and characterization of an esterase belonging to a new family via isolation from a metagenomic library of paper mill sludge.

A new bacterial lipolytic enzyme Est903 was obtained from paper mill sludge via metagenomic approach. Est903 displayed moderate similarities to two lipolytic enzymes from Rhodopirellula islandica and contained a distinctive pentapeptide motif (GFSAG) that differed from those of all known fourteen families of bacterial lipolytic enzymes. Est903 was regarded as from a new bacterial lipolytic enzyme family through multiple sequence alignment and phylogenetic analysis. The recombinant Est903 showed the highest activity for ρ-nitrophenol butyrate. The pH optimum and temperature optimum of the recombinant enzyme was 9.0 and 51 °C, respectively. Also, this enzyme displayed moderate thermostability, high activity under alkaline conditions, and good tolerance against several organic solvents. In addition, the compatibility test and washing performance analysis revealed that Est903 had good compatibility with commercial laundry detergent and high cleaning ability of oil stains. These good properties make Est903 a potential candidate in organic synthesis or detergent industry.

Elucidating the substrate specificities of acyl-lipid thioesterases from diverse plant taxa.

Acyl-ACP thioesterase enzymes, which cleave fatty acyl thioester bonds to release free fatty acids, contribute to much of the fatty acid diversity in plants. In Arabidopsis thaliana, a family of four single hot-dog fold domain, plastid-localized acyl-lipid thioesterases (AtALT1-4) generate medium-chain (C6-C14) fatty and ß-keto fatty acids as secondary metabolites. These volatile products may serve to attract insect pollinators or deter predatory insects. Homologs of AtALT1-4 are present in all plant taxa, but are nearly all uncharacterized. Despite high sequence identity, AtALT1-4 generate different lipid products, suggesting that ALT homologs in other plants also have highly varied activities. We investigated the catalytic diversity of ALT-like thioesterases by screening the substrate specificities of 15 ALT homologs from monocots, eudicots, a lycophyte, a green microalga, and the ancient gymnosperm Gingko biloba, via expression in Escherichia coli. Overall, these enzymes had highly varied substrate preferences compared to one another and to AtALT1-4, and could be classified into four catalytic groups comprising members from diverse taxa. Group 1 ALTs primarily generated 14:1 ß-keto fatty acids, Group 2 ALTs produced 6-10 carbon fatty/ß-keto fatty acids, Group 3 ALTs predominantly produced 12-14 carbon fatty acids, and Group 4 ALTs mainly generated 16 carbon fatty acids. Enzymes in each group differed significantly in the quantities of lipids and types of minor products they generated in E. coli. Medium-chain fatty acids are used to manufacture insecticides, pharmaceuticals, and biofuels, and ALT-like proteins are ideal candidates for metabolic engineering to produce specific fatty acids in significant quantities.

A Novel, Molybdenum-Containing Methionine Sulfoxide Reductase Supports Survival of Haemophilus influenzae in an In vivo Model of Infection.

Haemophilus influenzae is a host adapted human mucosal pathogen involved in a variety of acute and chronic respiratory tract infections, including chronic obstructive pulmonary disease and asthma, all of which rely on its ability to efficiently establish continuing interactions with the host. Here we report the characterization of a novel molybdenum enzyme, TorZ/MtsZ that supports interactions of H. influenzae with host cells during growth in oxygen-limited environments. Strains lacking TorZ/MtsZ showed a reduced ability to survive in contact with epithelial cells as shown by immunofluorescence microscopy and adherence/invasion assays. This included a reduction in the ability of the strain to invade human epithelial cells, a trait that could be linked to the persistence of H. influenzae. The observation that in a murine model of H. influenzae infection, strains lacking TorZ/MtsZ were almost undetectable after 72 h of infection, while ∼3.6 × 103 CFU/mL of the wild type strain were measured under the same conditions is consistent with this view. To understand how TorZ/MtsZ mediates this effect we purified and characterized the enzyme, and were able to show that it is an S- and N-oxide reductase with a stereospecificity for S-sulfoxides. The enzyme converts two physiologically relevant sulfoxides, biotin sulfoxide and methionine sulfoxide (MetSO), with the kinetic parameters suggesting that MetSO is the natural substrate of this enzyme. TorZ/MtsZ was unable to repair sulfoxides in oxidized Calmodulin, suggesting that a role in cell metabolism/energy generation and not protein repair is the key function of this enzyme. Phylogenetic analyses showed that H. influenzae TorZ/MtsZ is only distantly related to the Escherichia coli TorZ TMAO reductase, but instead is a representative of a new, previously uncharacterized clade of molybdenum enzyme that is widely distributed within the Pasteurellaceae family of pathogenic bacteria. It is likely that MtsZ/TorZ has a similar role in supporting host/pathogen interactions in other members of the Pasteurellaceae, which includes both human and animal pathogens.