A novel alkane monooxygenase (alkB) clade revealed by massive genomic survey and its dissemination association with IS elements.

Background: Alkanes are important components of fossil energy, such as crude oil. The alkane monooxygenase encoded by alkB gene performs the initial step of alkane degradation under aerobic conditions. The alkB gene is well studied due to its ubiquity as well as the availability of experimentally functional evidence. The alkBFGHJKL and alkST clusters are special kind of alkB-type alkane hydroxylase system, which encode all proteins necessary for converting alkanes into corresponding fatty acids. Methods: To explore whether the alkBFGHJKL and alkST clusters were widely distributed, we performed a large-scale analysis of isolate and metagenome assembled genome data (>390,000 genomes) to identify these clusters, together with distributions of corresponding taxonomy and niches. The set of alk-genes (including but not limited to alkBGHJ) located near each other on a DNA sequence was defined as an alk-gene cluster in this study. The alkB genes with alkGHJ located nearby on a DNA sequence were picked up for the investigation of putative alk-clusters. Results: A total of 120 alk-gene clusters were found in 117 genomes. All the 117 genomes are from strains located only in α- and γ-proteobacteria. The alkB genes located in alk-gene sets were clustered into a deeply branched mono-clade. Further analysis showed similarity organization types of alk-genes were observed within closely related species. Although a large number of IS elements were observed nearby, they did not lead to the wide spread of the alk-gene cluster. The uneven distribution of these elements indicated that there might be other factors affecting the transmission of alk-gene clusters. Conclusions: We conducted systematic bioinformatics research on alk-genes located near each other on a DNA sequence. This benchmark dataset of alk-genes can provide base line for exploring its evolutional and ecological importance in future studies.

A Glycolipid Glycosyltransferase with Broad Substrate Specificity from the Marine Bacterium "Candidatus Pelagibacter sp." Strain HTCC7211.

In the marine environment, phosphorus availability significantly affects the lipid composition in many cosmopolitan marine heterotrophic bacteria, including members of the SAR11 clade and the Roseobacter clade. Under phosphorus stress conditions, nonphosphorus sugar-containing glycoglycerolipids are substitutes for phospholipids in these bacteria. Although these glycoglycerolipids play an important role as surrogates for phospholipids under phosphate deprivation, glycoglycerolipid synthases in marine microbes are poorly studied. In the present study, we biochemically characterized a glycolipid glycosyltransferase (GTcp) from the marine bacterium "Candidatus Pelagibacter sp." strain HTCC7211, a member of the SAR11 clade. Our results showed that GTcp is able to act as a multifunctional enzyme by synthesizing different glycoglycerolipids with UDP-glucose, UDP-galactose, or UDP-glucuronic acid as sugar donors and diacylglycerol (DAG) as the acceptor. Analyses of enzyme kinetic parameters demonstrated that Mg2+ notably changes the enzyme's affinity for UDP-glucose, which improves its catalytic efficiency. Homology modeling and mutational analyses revealed binding sites for the sugar donor and the diacylglycerol lipid acceptor, which provided insights into the retaining mechanism of GTcp with its GT-B fold. A phylogenetic analysis showed that GTcp and its homologs form a group in the GT4 glycosyltransferase family. These results not only provide new insights into the glycoglycerolipid synthesis mechanism in lipid remodeling but also describe an efficient enzymatic tool for the future synthesis of bioactive molecules. IMPORTANCE The bilayer formed by membrane lipids serves as the containment unit for living microbial cells. In the marine environment, it has been firmly established that phytoplankton and heterotrophic bacteria can replace phospholipids with nonphosphorus sugar-containing glycoglycerolipids in response to phosphorus limitation. However, little is known about how these glycoglycerolipids are synthesized. Here, we determined the biochemical characteristics of a glycolipid glycosyltransferase (GTcp) from the marine bacterium "Candidatus Pelagibacter sp." strain HTCC7211. GTcp and its homologs form a group in the GT4 glycosyltransferase family and can synthesize neutral glycolipids (monoglucosyl-1,2-diacyl-sn-glycerol [MGlc-DAG] and monogalactosyl [MGal]-DAG) and monoglucuronic acid diacylglycerol (MGlcA-DAG). We also uncovered the key residues for DAG binding through molecular docking, site-direct mutagenesis, and subsequent enzyme activity assays. Our data provide new insights into the glycoglycerolipid synthesis mechanism in lipid remodeling.

Isolation and Characterization of an Acidic, Salt-Tolerant Endoglucanase Cel5A from a Bacterial Strain Martelella endophytica YC6887 Genome.

A Martelella endophytica (M. endophytica) strain YC6887 was previously isolated from the roots of a halophyte, Rosa rugosa, which was sequenced and characterized. The genomic and proteomic analysis showed a carbohydrate-degrading enzyme, endoglucanase Cel5A which was further characterized. The protein analysis revealed that this endoglucanase belongs to glycosidic hydrolase family 5 (GH5) with catalytic domain. This gene encodes 349-residue polypeptide and shows closest similarity with cellulases of other Martelella species. The protein was purified to homogeneity and shown that it was a 39 kDa protein. The purified recombinant Cel5A endoglucanase exhibited maximum activity at 50 °C and pH 4.5. The enzyme was salt tolerant and retained more than 50% residual activity up to 15% NaCl. The homology model structure of Cel5A displayed that it is stable and compact protein structure consisting of eleven α-helical structures and eight ß-sheets. According to the predicted ligand binding site after superimposition with Pseudomonas stutzeri endoglucanase Cel5A (PDB ID: 4LX4), it consisted of five amino acid Asn157, Tyr116, Glu158, Glu270 and Trp303 that may be the expected active site of Cel5A from YC6887. This presented that our strain M. endophytica YC6887 that produces cellulase partially degrade the insoluble polysaccharides into reducing sugars.

Improving the enantioselectivity of halohydrin dehalogenase for the synthesis of (R)-benzyl glycidyl ether via biocatalytic azidolysis.

Halohydrin dehalogenases (HHDHs) are valuable biocatalysts for the synthesis of enantiopure benzyl glycidyl ether (BGE) and its derivatives, which are important synthetic intermediates for anti-cancer and anti-obesity drugs. However, all the reported HHDHs exhibit low enantioselectivity. In this study, we screened site-saturation mutagenesis libraries of AbHHDH at positions R89, A136, V137, P178, N179, F180, I181, Y186 and F187 for mutants with enhanced enantioselectivity toward BGE. The four improved variant R89V, R89Y, R89K and V137I were identified, and the double mutant R89Y/V137I showed 2.9-fold higher enantioselectivity than the wild type. The regions of HHDH containing the identified mutations were analyzed by homology modeling to explain the changes of enantioselectivity. Kinetic resolution of 20 to 100 mM BGE using whole cells of Escherichia coli expressing the mutant R89Y/V137I resulted in (R)-BGE yields of 42 to 32.5%, with ee >99%. This study improves our understanding of the enantioselectivity of HHDHs and contributes improved biocatalysts for the kinetic resolution of BGE.

α-proteobacteria synthesize biotin precursor pimeloyl-ACP using BioZ 3-ketoacyl-ACP synthase and lysine catabolism.

Pimelic acid, a seven carbon α,ω-dicarboxylic acid (heptanedioic acid), is known to provide seven of the ten biotin carbon atoms including all those of the valeryl side chain. Distinct pimelate synthesis pathways were recently elucidated in Escherichia coli and Bacillus subtilis where fatty acid synthesis plus dedicated biotin enzymes produce the pimelate moiety. In contrast, the α-proteobacteria which include important plant and mammalian pathogens plus plant symbionts, lack all of the known pimelate synthesis genes and instead encode bioZ genes. Here we report a pathway in which BioZ proteins catalyze a 3-ketoacyl-acyl carrier protein (ACP) synthase III-like reaction to produce pimeloyl-ACP with five of the seven pimelate carbon atoms being derived from glutaryl-CoA, an intermediate in lysine degradation. Agrobacterium tumefaciens strains either deleted for bioZ or which encode a BioZ active site mutant are biotin auxotrophs, as are strains defective in CaiB which catalyzes glutaryl-CoA synthesis from glutarate and succinyl-CoA.

Draft genome sequence of Parvularcula flava strain NH6-79 T, revealing its role as a cellulolytic enzymes producer.

To date, the genus Parvularcula consists of 6 species and no potential application of this genus was reported. Current study presents the genome sequence of Parvularcula flava strain NH6-79 T and its cellulolytic enzyme analysis. The assembled draft genome of strain NH6-79 T consists of 9 contigs and 7 scaffolds with 3.68 Mbp in size and GC content of 59.87%. From a total of 3,465 genes predicted, 96 of them are annotated as glycoside hydrolases (GHs). Within these GHs, 20 encoded genes are related to cellulosic biomass degradation, including 12 endoglucanases (5 GH10, 4 GH5, and 3 GH51), 2 exoglucanases (GH9) and 6 ß-glucosidases (GH3). In addition, highest relative enzyme activities (endoglucanase, exoglucanase, and ß-glucosidase) were observed at 27th hour when the strain was cultured in the carboxymethyl cellulose/Avicel®-containing medium for 45 h. The combination of genome analysis with experimental studies indicated the ability of strain NH6-79 T to produce extracellular endoglucanase, exoglucanase, and ß-glucosidase. These findings suggest the potential of Parvularcula flava strain NH6-79 T in cellulose-containing biomass degradation and that the strain could be used in cellulosic biorefining process.

Response of particle-associated bacteria to long-term heavy metal contamination in a tropical estuary.

Estuaries being the connecting link between terrestrial and marine environment, experience spatial variations in the hydrographic variables as well as concentrations of pollutants. The present study reports a contrasting difference in the metal tolerance and enzyme activity of particle-associated bacteria (PAB) isolated from the upstream and downstream reaches of a tropical estuary [Cochin Estuary (CE) in the southwest coast of India], exposed to different levels of heavy metal contamination. The upstream of the estuary has been overloaded with heavy metals in the last few decades, while the downstream is less polluted. There were only 25% of culturable PAB phylogenetically common in both upstream and downstream. The PAB isolated from the upstream were dominated by γ-proteobacteria (48.1%) followed by α-proteobacteria (25.0%), while it was in the reverse order of α-proteobacteria (45.9%) and γ-proteobacteria (36.1%) in the downstream. More number of PAB from the upstream showed tolerance to higher concentrations of Zn and Cd. The Acinetobacter sp. MMRF1051 isolated from the upstream showed tolerance up to 250 mM Zn, 100 mM Cd, and 250 mM Ni. The enzyme expression profile of PAB from downstream was in the order of lipase > phosphatase > ß-glucosidase > aminopeptidase, while it was in the order of ß-glucosidase > lipase > aminopeptidase > phosphatase in the upstream of the estuary. The present study shows the selective pressure exerted by heavy metal pollution on the diversity of culturable bacteria associated with particulate matter in a tropical estuary. Also, the variation in their enzyme activities may impinge the remineralization of particulate organic matter (POM) in the system and may impart adverse impacts on ecosystem functioning.

Significant improvement of the enantioselectivity of a halohydrin dehalogenase for asymmetric epoxide ring opening reactions by protein engineering.

Halohydrin dehalogenases (HHDHs) have attracted much attention due to their ability to synthesize enantiomerically enriched epoxides and ß-haloalcohols. However, most of the HHDHs exhibit low enantioselectivity. Here, a HHDH from the alphaproteobacteria isolate 46_93_T64 (AbHHDH), which shows only poor enantioselectivity in the catalytic resolution of rac-PGE (E = 9.9), has been subjected to protein engineering to enhance its enantioselectivity. Eight mutants (R89K, R89Y, V137I, P178A, N179Q, N179L, F187L, F187A) showed better enantioselectivity than the wild type. The best single mutant N179L (E = 93.0) showed a remarkable 9.4-fold increase in the enantioselectivity. Then, the single mutations were combined to produce the double, triple, quadruple, and quintuple mutants. Among the combinational mutants, the best variant (R89Y/N179L) showed an increased E value of up to 48. The E values of the variants N179L and R89Y/N179L for other epoxides 2-7 were 12.2 to > 200, which showed great improvement compared to 1.2 to 10.5 for the wild type. Using the variant N179L, enantiopure (R)-PGE with > 99% ee could be readily prepared, affording a high yield and a high concentration.

Structural insight into bi-functional malonyl-CoA reductase.

The bi-functional malonyl-CoA reductase is a key enzyme of the 3-hydroxypropionate bi-cycle for bacterial CO2 fixation, catalysing the reduction of malonyl-CoA to malonate semialdehyde and further reduction to 3-hydroxypropionate. Here, we report the crystal structure and the full-length architecture of malonyl-CoA reductase from Porphyrobacter dokdonensis. The malonyl-CoA reductase monomer of 1230 amino acids consists of four tandemly arranged short-chain dehydrogenases/reductases, with two catalytic and two non-catalytic short-chain dehydrogenases/reductases, and forms a homodimer through paring contact of two malonyl-CoA reductase monomers. The complex structures with its cofactors and substrates revealed that the malonyl-CoA substrate site is formed by the cooperation of two short-chain dehydrogenases/reductases and one novel extra domain, while only one catalytic short-chain dehydrogenase/reductase contributes to the formation of the malonic semialdehyde-binding site. The phylogenetic and structural analyses also suggest that the bacterial bi-functional malonyl-CoA has a structural origin that is completely different from the archaeal mono-functional malonyl-CoA and malonic semialdehyde reductase, and thereby constitute an efficient enzyme.

Light-driven carbon-carbon bond formation via CO2 reduction catalyzed by complexes of CdS nanorods and a 2-oxoacid oxidoreductase.

Redox enzymes are capable of catalyzing a vast array of useful reactions, but they require redox partners that donate or accept electrons. Semiconductor nanocrystals provide a mechanism to convert absorbed photon energy into redox equivalents for enzyme catalysis. Here, we describe a system for photochemical carbon-carbon bond formation to make 2-oxoglutarate by coupling CO2 with a succinyl group. Photoexcited electrons from cadmium sulfide nanorods (CdS NRs) transfer to 2-oxoglutarate:ferredoxin oxidoreductase from Magnetococcus marinus MC-1 (MmOGOR), which catalyzes a carbon-carbon bond formation reaction. We thereby decouple MmOGOR from its native role in the reductive tricarboxylic acid cycle and drive it directly with light. We examine the dependence of 2-oxoglutarate formation on a variety of factors and, using ultrafast transient absorption spectroscopy, elucidate the critical role of electron transfer (ET) from CdS NRs to MmOGOR. We find that the efficiency of this ET depends strongly on whether the succinyl CoA (SCoA) cosubstrate is bound at the MmOGOR active site. We hypothesize that the conformational changes due to SCoA binding impact the CdS NR-MmOGOR interaction in a manner that decreases ET efficiency compared to the enzyme with no cosubstrate bound. Our work reveals structural considerations for the nano-bio interfaces involved in light-driven enzyme catalysis and points to the competing factors of enzyme catalysis and ET efficiency that may arise when complex enzyme reactions are driven by artificial light absorbers.

Biomimetic mineralization of nitrile hydratase into a mesoporous cobalt-based metal-organic framework for efficient biocatalysis.

Nitrile hydratases (NHases) have attracted considerable attention owing to their application in the synthesis of valuable amides under mild conditions. However, the poor stability of NHases is still one of the main drawbacks for their industrial application. Recently, mesoporous metal-organic frameworks (MOFs) have been explored as an attractive support material for immobilizing enzymes. Here, we encapsulated a recombinant cobalt-type NHase from Aurantimonas manganoxydans into the cobalt-based MOF ZIF-67 by a biomimetic mineralization strategy. The nano-catalyst NHase1229@ZIF-67 shows high catalytic activity for the hydration of 3-cyanopyridine to nicotinamide, and its specific activity reached 29.5 U mg-1. The NHase1229@ZIF-67 nanoparticles show a significant improvement in the thermal stability of NHase1229. The optimum reaction temperature of NHase1229@ZIF-67 is at 50-55 °C, and it still retained 40% of the maximum activity at 70 °C. However, the free NHase1229 completely lost its catalytic activity at 70 °C. The half-lives of NHase1229@ZIF-67 at 30 and 40 °C were 102.0 h and 26.5 h, respectively. NHase1229@ZIF-67 nanoparticles exhibit an excellent cycling performance, and their catalytic efficiency did not significantly decrease in the initial 6 cycles using 0.9 M 3-cyanopyridine as the substrate. In a fed-batch reaction, NHase1229@ZIF-67 can efficiently hydrate 3-cyanopyridine to nicotinamide, and the space-time yield was calculated to be 110 g·L-1·h-1. Therefore, the cobalt-type NHase was immobilized in MOF ZIF-67, which is shown as a potential nanocatalyst for the large-scale industrial preparation of nicotinamide.

Database Mining for Novel Bacterial ß-Etherases, Glutathione-Dependent Lignin-Degrading Enzymes.

Lignin is the most abundant aromatic polymer in nature and a promising renewable source for the provision of aromatic platform chemicals and biofuels. ß-Etherases are enzymes with a promising potential for application in lignin depolymerization due to their selectivity in the cleavage of ß-O-4 aryl ether bonds. However, only a very limited number of these enzymes have been described and characterized so far. Using peptide pattern recognition (PPR) as well as phylogenetic analyses, 96 putatively novel ß-etherases have been identified, some even originating from bacteria outside the order Sphingomonadales A set of 13 diverse enzymes was selected for biochemical characterization, and ß-etherase activity was confirmed for all of them. Some enzymes displayed up to 3-fold higher activity than previously known ß-etherases. Moreover, conserved sequence motifs specific for either LigE- or LigF-type enzymes were deduced from multiple-sequence alignments and the PPR-derived peptides. In combination with structural information available for the ß-etherases LigE and LigF, insight into the potential structural and/or functional role of conserved residues within these sequence motifs is provided. Phylogenetic analyses further suggest the presence of additional bacterial enzymes with potential ß-etherase activity outside the classical LigE- and LigF-type enzymes as well as the recently described heterodimeric ß-etherases.IMPORTANCE The use of biomass as a renewable source and replacement for crude oil for the provision of chemicals and fuels is of major importance for current and future societies. Lignin, the most abundant aromatic polymer in nature, holds promise as a renewable starting material for the generation of required aromatic structures. However, a controlled and selective lignin depolymerization to yield desired aromatic structures is a very challenging task. In this regard, bacterial ß-etherases are especially interesting, as they are able to cleave the most abundant bond type in lignin with high selectivity. With this study, we significantly expanded the toolbox of available ß-etherases for application in lignin depolymerization and discovered more active as well as diverse enzymes than previously known. Moreover, the identification of further ß-etherases by sequence database mining in the future will be facilitated considerably through our deduced etherase-specific sequence motifs.

Cloning and characterization of an oxiranedicarboxylate hydrolase from Labrys sp. WH-1.

OBJECTIVE: This study aimed to clone and characterize the oxiranedicarboxylate hydrolase (ORCH) from Labrys sp. WH-1. METHODS: Purification by column chromatography, characterization of enzymatic properties, gene cloning by protein terminal sequencing and polymerase chain reaction (PCR), and sequence analysis by secondary structure prediction and multiple sequence alignment were performed. RESULTS: The ORCH from Labrys sp. WH-1 was purified 26-fold with a yield of 12.7%. It is a monomer with an isoelectric point (pI) of 8.57 and molecular mass of 30.2 kDa. It was stable up to 55 °C with temperature at which the activity of the enzyme decreased by 50% in 15 min (T5015) of 61 °C and the half-life at 50 °C (t1/2, 50 °C) of 51 min and was also stable from pH 4 to 10, with maximum activity at 55 °C and pH 8.5. It is a metal-independent enzyme and strongly inhibited by Cu2+, Ag+, and anionic surfactants. Its kinetic parameters (Km, kcat, and kcat/Km) were 18.7 mmol/L, 222.3 s-1, and 11.9 mmol/(L·s), respectively. The ORCH gene, which contained an open reading frame (ORF) of 825 bp encoding 274 amino acid residues, was overexpressed in Escherichia coli and the enzyme activity was 33 times higher than that of the wild strain. CONCLUSIONS: The catalytic efficiency and thermal stability of the ORCH from Labrys sp. WH-1 were the best among the reported ORCHs, and it provides an alternative catalyst for preparation of L(+)-2,3-dihydrobutanedioic acid.

A two-component protease in Methylorubrum extorquens with high activity toward the peptide precursor of the redox cofactor pyrroloquinoline quinone.

Pyrroloquinoline quinone is a prominent redox cofactor in many prokaryotes, produced from a ribosomally synthesized and post-translationally modified peptide PqqA via a pathway comprising four conserved proteins PqqB-E. These four proteins are now fairly well-characterized and span radical SAM activity (PqqE), aided by a peptide chaperone (PqqD), a dual hydroxylase (PqqB), and an eight-electron, eight-proton oxidase (PqqC). A full description of this pathway has been hampered by a lack of information regarding a protease/peptidase required for the excision of an early, cross-linked di-amino acid precursor to pyrroloquinoline quinone. Herein, we isolated and characterized a two-component heterodimer protein from the α-proteobacterium Methylobacterium (Methylorubrum) extorquens that can rapidly catalyze cleavage of PqqA into smaller peptides. Using pulldown assays, surface plasmon resonance, and isothermal calorimetry, we demonstrated the formation of a complex PqqF/PqqG, with a KD of 300 nm We created a molecular model of the heterodimer by comparison with the Sphingomonas sp. A1 M16B Sph2681/Sph2682 protease. Analysis of time-dependent patterns for the appearance of proteolysis products indicates high specificity of PqqF/PqqG for serine side chains. We hypothesize that PqqF/PqqG initially cleaves between the PqqE/PqqD-generated cross-linked form of PqqA, with nonspecific cellular proteases completing the release of a suitable substrate for the downstream enzyme PqqB. The finding of a protease that specifically targets serine side chains is rare, and we propose that this activity may be useful in proteomic analyses of the large family of proteins that have undergone post-translational phosphorylation at serine.

Characterization of the Propham Biodegradation Pathway in Starkeya sp. Strain YW6 and Cloning of a Novel Amidase Gene mmH.

We previously isolated a monocrotophos-degrading strain Starkeya sp. YW6, which could also degrade propham. Here, we show that strain YW6 metabolizes propham via a pathway in which propham is initially hydrolyzed to aniline and then converted to catechol, which is then oxidized via an ortho-cleavage pathway. The novel amidase gene mmH was cloned from strain YW6 and expressed in Escherichia coli BL21(DE3). MmH, which exhibits aryl acylamidase activity, was purified for enzymatic analysis. Bioinformatic analysis confirmed that MmH belongs to the amidase signature (AS) enzyme family and shares 26-50% identity with several AS family members. MmH (molecular mass of 53 kDa) was most active at 40 °C and pH 8.0 and showed high activity toward propham, with Kcat and Km values of 33.4 s-1 and 16.9 µM, respectively. These characteristics make MmH suitable for novel amide biosynthesis and environmental remediation.

A novel SGNH family hydrolase Ali5 with thioesterase activity and a GNSL motif but without a classic GDSL motif from Altererythrobacter ishigakiensis.

OBJECTIVE: We aimed to characterize a novel SGNH (Ser-Gly-Asn-His) family hydrolase from the annotated genome of marine bacteria with new features. RESULTS: A novel esterase Ali5 from Altererythrobacter ishigakiensis has been identified and classified into SGNH family. Ali5 presented a novel GNSL (Gly-Asn-Ser-Leu(X)) motif that differs from the classic GDSL (Gly-Asp-Ser-Leu(X)) motif of SGNH family. The enzyme has esterase and thioesterase activity and exhibited apparent temperature and pH optima of 40 °C and pH 7.5 (in phosphate buffer), respectively. Ali5 was found to be halotolerant and thermostable, and exhibited strong resistance to several organic solvents and metal ions. The residue Tyr196 has a great influence on the catalytic activity, which was proved by site-directed mutagenesis and subsequent kinetic characterization. CONCLUSION: The esterase Ali5 with esterase and thioesterase activities, salt and metal ions resistance and unique structural features was identified, which holds promise for research on the SGNH family of hydrolases.

Efficient Production of 2,6-Difluorobenzamide by Recombinant Escherichia coli Expressing the Aurantimonas manganoxydans Nitrile Hydratase.

2,6-Difluorobenzamide is an important intermediate with many applications in pesticide industries. Through screening a library of recombinant nitrile hydratases, the nitrile hydratase from Aurantimonas manganoxydans ATCC BAA-1229 was selected for production of 2,6-difluorobenzamide from 2,6-difluorobenzonitrile. Key parameters of the biocatalytic process, including temperature, pH, substrate loading, and substrate feeding mode, were optimized. Finally, 314 g/L of 2,6-difluorobenzamide was produced in a simple batch process within 11 h without formation of any by-product in an economical non-buffer system and similar result was obtained when scaled up to 30 L. This study constitutes the first report of 2,6-difluorobenzamide significant production using a recombinant Escherichia coli-based biocatalyst.

Cloning and characterization of an oxiranedicarboxylate hydrolase from Labrys sp. WH-1 / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

OBJECTIVE@#This study aimed to clone and characterize the oxiranedicarboxylate hydrolase (ORCH) from Labrys sp. WH-1.@*METHODS@#Purification by column chromatography, characterization of enzymatic properties, gene cloning by protein terminal sequencing and polymerase chain reaction (PCR), and sequence analysis by secondary structure prediction and multiple sequence alignment were performed.@*RESULTS@#The ORCH from Labrys sp. WH-1 was purified 26-fold with a yield of 12.7%. It is a monomer with an isoelectric point (pI) of 8.57 and molecular mass of 30.2 kDa. It was stable up to 55 °C with temperature at which the activity of the enzyme decreased by 50% in 15 min (T5015) of 61 °C and the half-life at 50 °C (t1/2, 50 °C) of 51 min and was also stable from pH 4 to 10, with maximum activity at 55 °C and pH 8.5. It is a metal-independent enzyme and strongly inhibited by Cu2+, Ag+, and anionic surfactants. Its kinetic parameters (Km, kcat, and kcat/Km) were 18.7 mmol/L, 222.3 s-1, and 11.9 mmol/(L·s), respectively. The ORCH gene, which contained an open reading frame (ORF) of 825 bp encoding 274 amino acid residues, was overexpressed in Escherichia coli and the enzyme activity was 33 times higher than that of the wild strain.@*CONCLUSIONS@#The catalytic efficiency and thermal stability of the ORCH from Labrys sp. WH-1 were the best among the reported ORCHs, and it provides an alternative catalyst for preparation of L(+)-2,3-dihydrobutanedioic acid.

N-terminal engineering of overlapping genes in the nitrile hydratase gene cluster improved its activity.

Nitrile hydratase which catalyzes the hydration of nitriles to the corresponding amides is operon-encoded. However, when heterologously expressed, genes in the same operon are usually not equally expressed, and the ratio needs to be fine-tuned. A gene cluster of three genes (corresponding to α-subunit, ß-subunit and activator) encoding the nitrile hydratase was cloned from Aurantimonas manganoxydans ATCC BAA-1229 and expressed in Escherichia coli. However, difficulty was encountered in heterologous expression of the activator and the expression level of ß-subunit was lower than that of α-subunit, which together resulted in low catalytic efficiency. To improve the expression of activator, a set of SKIK tags were fused to the N-terminus of the activator. To elevate the expression level of ß-subunit, a silent mutation strategy was applied in the overlapping sequence with α-subunit around its translation initial region. Finally, the expression of ß-subunit and activator were improved and the maximum activity of NHase1229 was doubled, reaching 160 U/mL towards 3-cyanopyridine. These results indicate that N-terminal engineering is an efficient strategy for optimizing the expression of multiple genes in operons.

Unidirectional regulation of the F1FO-ATP synthase nanomotor by the ζ pawl-ratchet inhibitor protein of Paracoccus denitrificans and related α-proteobacteria.

The ATP synthase is a reversible nanomotor that gyrates its central rotor clockwise (CW) to synthesize ATP and in counter clockwise (CCW) direction to hydrolyse it. In bacteria and mitochondria, two natural inhibitor proteins, namely the ε and IF1 subunits, prevent the wasteful CCW F1FO-ATPase activity by blocking γ rotation at the αDP/ßDP/γ interface of the F1 portion. In Paracoccus denitrificans and related α-proteobacteria, we discovered a different natural F1-ATPase inhibitor named ζ. Here we revise the functional and structural data showing that this novel ζ subunit, although being different to ε and IF1, it also binds to the αDP/ßDP/γ interface of the F1 of P. denitrificans. ζ shifts its N-terminal inhibitory domain from an intrinsically disordered protein region (IDPr) to an α-helix when inserted in the αDP/ßDP/γ interface. We showed for the first time the key role of a natural ATP synthase inhibitor by the distinctive phenotype of a Δζ knockout mutant in P. denitrificans. ζ blocks exclusively the CCW F1FO-ATPase rotation without affecting the CW-F1FO-ATP synthase turnover, confirming that ζ is important for respiratory bacterial growth by working as a unidirectional pawl-ratchet PdF1FO-ATPase inhibitor, thus preventing the wasteful consumption of cellular ATP. In summary, ζ is a useful model that mimics mitochondrial IF1 but in α-proteobacteria. The structural, functional, and endosymbiotic evolutionary implications of this ζ inhibitor are discussed to shed light on the natural control mechanisms of the three natural inhibitor proteins (ε, ζ, and IF1) of this unique ATP synthase nanomotor, essential for life.