Transition States for Psychrophilic and Mesophilic (R)-3-Hydroxybutyrate Dehydrogenase-Catalyzed Hydride Transfer at Sub-zero Temperatures.

(R)-3-Hydroxybutyrate dehydrogenase (HBDH) catalyzes the NADH-dependent reduction of 3-oxocarboxylates to (R)-3-hydroxycarboxylates. The active sites of a pair of cold- and warm-adapted HBDHs are identical except for a single residue, yet kinetics evaluated at -5, 0, and 5 °C show a much higher steady-state rate constant (kcat) for the cold-adapted than for the warm-adapted HBDH. Intriguingly, single-turnover rate constants (kSTO) are strikingly similar between the two orthologues. Psychrophilic HBDH primary deuterium kinetic isotope effects on kcat (Dkcat) and kSTO (DkSTO) decrease at lower temperatures, suggesting more efficient hydride transfer relative to other steps as the temperature decreases. However, mesophilic HBDH Dkcat and DkSTO are generally temperature-independent. The DkSTO data allowed calculation of intrinsic primary deuterium kinetic isotope effects. Intrinsic isotope effects of 4.2 and 3.9 for cold- and warm-adapted HBDH, respectively, at 5 °C, supported by quantum mechanics/molecular mechanics calculations, point to a late transition state for both orthologues. Conversely, intrinsic isotope effects of 5.7 and 3.1 for cold- and warm-adapted HBDH, respectively, at -5 °C indicate the transition state becomes nearly symmetric for the psychrophilic enzyme, but more asymmetric for the mesophilic enzyme. His-to-Asn and Asn-to-His mutations in the psychrophilic and mesophilic HBDH active sites, respectively, swap the single active-site position where these orthologues diverge. At 5 °C, the His-to-Asn mutation in psychrophilic HBDH decreases Dkcat to 3.1, suggesting a decrease in transition-state symmetry, while the His-to-Asn mutation in mesophilic HBDH increases Dkcat to 4.4, indicating an increase in transition-state symmetry. Hence, temperature adaptation and a single divergent active-site residue may influence transition-state geometry in HBDHs.

NADP+ -dependent isocitrate dehydrogenase isozymes from a psychrotrophic bacterium, Psychrobacter sp. strain 13A.

The genes encoding dimeric and monomeric isocitrate dehydrogenase (IDH) isozymes from a psychrotrophic bacterium, strain 13A (13AIDH-D and 13AIDH-M, respectively), were cloned and sequenced. The deduced amino acid sequences of these two IDHs showed high degrees of identity with those of bacteria of genus Psychrobacter. Analysis of the 16S ribosomal RNA gene of the strain 13A revealed that this bacterium is classified to genus Psychrobacter. The optimum temperatures for activities of 13AIDH-D and 13AIDH-M were 55°C and 45°C, respectively, indicating that they are mesophilic. On the contrary, 13AIDH-D maintained 90% of its maximum activity after incubation for 10 min at 50°C, while the 13AIDH-M activity was completely lost under the same condition. In addition, 13AIDH-D showed much higher specific activity than 13AIDH-M. From northern and western blot analyses, the 13AIDH-D gene was found to be not transcribed under the growth conditions tested in this study. However, the catalytic ability of the mesophilic 13AIDH-M was concluded to be enough to sustain the growth of strain 13A at low temperatures. Therefore, a novel pattern of the contribution of IDH isozymes in cold-living bacteria to their growth at low temperatures was confirmed in strain 13A.

A novel ATP dependent dimethylsulfoniopropionate lyase in bacteria that releases dimethyl sulfide and acryloyl-CoA.

Dimethylsulfoniopropionate (DMSP) is an abundant and ubiquitous organosulfur molecule in marine environments with important roles in global sulfur and nutrient cycling. Diverse DMSP lyases in some algae, bacteria, and fungi cleave DMSP to yield gaseous dimethyl sulfide (DMS), an infochemical with important roles in atmospheric chemistry. Here, we identified a novel ATP-dependent DMSP lyase, DddX. DddX belongs to the acyl-CoA synthetase superfamily and is distinct from the eight other known DMSP lyases. DddX catalyses the conversion of DMSP to DMS via a two-step reaction: the ligation of DMSP with CoA to form the intermediate DMSP-CoA, which is then cleaved to DMS and acryloyl-CoA. The novel catalytic mechanism was elucidated by structural and biochemical analyses. DddX is found in several Alphaproteobacteria, Gammaproteobacteria, and Firmicutes, suggesting that this new DMSP lyase may play an overlooked role in DMSP/DMS cycles.

The global sulfur cycle is a collection of geological and biological processes that circulate sulfur-containing compounds through the oceans, rocks and atmosphere. Sulfur itself is essential for life and important for plant growth, hence its widespread use in fertilizers. Marine organisms such as bacteria, algae and phytoplankton produce one particular sulfur compound, called dimethylsulfoniopropionate, or DMSP, in massive amounts. DMSP made in the oceans gets readily converted into a gas called dimethyl sulfide (DMS), which is the largest natural source of sulfur entering the atmosphere. In the air, DMS is converted to sulfate and other by-products that can act as cloud condensation nuclei, which, as the name suggests, are involved in cloud formation. In this way, DMS can influence weather and climate, so it is often referred to as 'climate-active' gas. At least eight enzymes are known to cleave DMSP into DMS gas with a few by-products. These enzymes are found in algae, bacteria and fungi, and are referred to as lyases, for the way they breakdown their target compounds (DMSP, in this case). Recently, researchers have identified some bacteria that produce DMS from DMSP without using known DMSP lyases. This suggests there are other, unidentified enzymes that act on DMSP in nature, and likely contribute to global sulfur cycling. Li, Wang et al. set out to uncover new enzymes responsible for converting the DMSP that marine bacteria produce into gaseous DMS. One new enzyme called DddX was identified and found to belong to a superfamily of enzymes quite separate to other known DMSP lyases. Li, Wang et al. also showed how DddX drives the conversion of DMSP to DMS in a two-step reaction, and that the enzyme is found across several classes of bacteria. Further experiments to characterise the protein structure of DddX also revealed the molecular mechanism for its catalytic action. This study offers important insights into how marine bacteria generate the climatically important gas DMS from DMSP, leading to a better understanding of the global sulfur cycle. It gives microbial ecologists a more comprehensive perspective of these environmental processes, and provides biochemists with data on a family of enzymes not previously known to act on sulfur-containing compounds.

Characterization of two enzymes from Psychrobacter cryohalolentis that are required for the biosynthesis of an unusual diacetamido-d-sugar.

Psychrobacter cryohalolentis strain K5T is a Gram-negative organism first isolated in 2006. It has a complex O-antigen that contains, in addition to l-rhamnose and d-galactose, two diacetamido- and a triacetamido-sugar. The biochemical pathways for the production of these unusual sugars are presently unknown. Utilizing the published genome sequence of the organism, we hypothesized that the genes 0612, 0638, and 0637 encode for a 4,6-dehydratase, an aminotransferase, and an N-acetyltransferase, respectively, which would be required for the biosynthesis of one of the diacetamido-sugars, 2,4-diacetamido-2,4,6-trideoxy-d-glucose, starting from UDP-N-acetylglucosamine. Here we present functional and structural data on the proteins encoded by the 0638 and 0637 genes. The kinetic properties of these enzymes were investigated by a discontinuous HPLC assay. An X-ray crystallographic structure of 0638, determined in its external aldimine form to 1.3 Å resolution, demonstrated the manner in which the UDP ligand is positioned into the active site. It is strikingly different from that previously observed for PglE from Campylobacter jejuni, which functions on the same substrate. Four X-ray crystallographic structures were also determined for 0637 in various complexed states at resolutions between 1.3 and 1.55 Å. Remarkably, a tetrahedral intermediate mimicking the presumed transition state was trapped in one of the complexes. The data presented herein confirm the hypothesized functions of these enzymes and provide new insight into an unusual sugar biosynthetic pathway in Gram-negative bacteria. We also describe an efficient method for acetyl-CoA synthesis that allowed us to overcome its prohibitive cost for this analysis.

A New Cold-Adapted and Salt-Tolerant Glutathione Reductase from Antarctic Psychrophilic Bacterium Psychrobacter sp. and Its Resistance to Oxidation.

A new glutathione reductase gene (psgr) coding for glutathione reductase (GR) from an Antarctic bacterium was cloned and overexpressed into Escherichia coli (E. coli). A sequence analysis revealed that PsGR is a protein consisting of 451 amino acids, and homology modeling demonstrated that PsGR has fewer hydrogen bonds and salt bridges, which might lead to improved conformational flexibility at low temperatures. PsGR possesses the flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) binding motifs. Recombinant PsGR (rPsGR) was purified using Ni-NTA affinity chromatography and was found to have a molecular mass of approximately 53.5 kDa. rPsGR was found to be optimally active at 25 °C and a pH of 7.5. It was found to be a cold-adapted enzyme, with approximately 42% of its optimal activity remaining at 0 °C. Moreover, rPsGR was most active in 1.0 M NaCl and 62.5% of its full activity remained in 3.0 M NaCl, demonstrating its high salt tolerance. Furthermore, rPsGR was found to have a higher substrate affinity for NADPH than for GSSG (oxidized glutathione). rPsGR provided protection against peroxide (H2O2)-induced oxidative stress in recombinant cells, and displayed potential application as an antioxidant protein. The results of the present study provide a sound basis for the study of the structural characteristics and catalytic characterization of cold-adapted GR.

Crystal structure of the reactive intermediate/imine deaminase A homolog from the Antarctic bacterium Psychrobacter sp. PAMC 21119.

The RidA subfamily proteins catalyze the deamination reaction of enamine/imine intermediates, which are metabolites of amino acids such as threonine and serine. Numerous structural and functional studies have been conducted on RidA isolated from mesophiles and thermophiles. However, little is known about the structure of the RidA proteins isolated from psychrophiles. In the present study, we elucidated the crystal structure of RidA from the Antarctic bacterium Psychrobacter sp. PAMC 21119 (Pp-RidA) at 1.6 Å resolution to identify the structural properties contributing to cold-adaptability. Although the overall structure of Pp-RidA is similar to those of its homologues, it exhibits specific structural arrangements of a loop positioned near the active site, which is assumed to play a role in covering the active site of catalysis. In addition, the surface electrostatic potential of Pp-RidA suggested that it exhibits stronger electrostatic distribution relative to its homologues. Our results provide novel insights into the key determinants of cold-adaptability.

A novel cold-adapted nitroreductase from Psychrobactersp. ANT206: Heterologous expression, characterization and nitrobenzene reduction capacity.

Widely used in multiple industrial processes, nitro-aromatic compounds, especially nitrobenzene, in low temperature environment are considered as heavy pollutants. Among the disposal methods, the bioreduction method has attracted much attention. In this study, a novel cold-adapted nitroreductase gene (psntr) was cloned from Antarctic sea-ice bacteria Psychrobacter sp. ANT206. The psntr gene was 813 bp in length and encoded a protein with flavin mononucleotide (FMN) binding sites. Homology modeling was performed to obtain structural information such as the longer loops and reduced amount of hydrogen bonds, which might be related to the high catalytic efficiency of PsNTR at low temperature. The psntr gene was successfully cloned in cold-shock pCold I vector and transformed to the expression host Escherichia coli (E. coli) BL21 with the induction by isopropyl ß-D-thiogalactoside (IPTG) at low temperature (16 °C) for 24 h. The recombinant PsNTR (rPsNTR) was purified using Ni-NTA with the specific activity of 51.59 µmol/min/mg. Interestingly, rPsNTR displayed the highest activity at 25 °C and still maintained 46.9% of the activity at 0 °C. rPsNTR also exhibited the highest activity (136.4%) at 1.0 M NaCl with incredible salt tolerance. The kinetic parameters and substrates specificity analysis demonstrated that rPsNTR could reduce various nitro-aromatic compounds. Moreover, the result of the reduction capability revealed that the recombinant E. coli exhibited a maximum nitrobenzene reduction rate of 3.03 mM/h at 16 °C. These findings revealed that the characteristics of rPsNTR might make it an excellent candidate for the bioreduction of various nitro-aromatic compounds in the low temperature and high-salt wastewater.

Crystal structure of the cold-adapted haloalkane dehalogenase DpcA from Psychrobacter cryohalolentis K5.

Haloalkane dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, haloalkane dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the haloalkane dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure-function relationships of cold-adapted enzymes.

Cloning, expression and enzymatic characteristics of a 2-Cys peroxiredoxin from Antarctic sea-ice bacterium Psychrobacter sp. ANT206.

Peroxiredoxin (Prx, EC 1.11.1.15) is a family of the thiol-dependent antioxidant enzyme. In this study, a cold-adapted Prx gene from Antarctic psychrophilic bacterium Psychrobacter sp. ANT206 (PsPrx) consisted of an open reading frame (ORF) of 567 bp was cloned. Amino acid sequence analysis revealed that PsPrx contained one catalytic site (Thr45, Cys48 and Arg121) and could be categorized as a typical 2-Cys Prx. Compared with the mesophilic StPrx, PsPrx with a reduced amount of hydrogen bonds and salt bridges and other characteristics, may be responsible for its enzymatic stability and flexibility at low temperature. The recombinant PsPrx (rPsPrx) was purified to homogeneity by Ni-NTA and its enzymatic characterization was described. Interestingly, rPsPrx exhibited the maximum activity at 30 °C and remained 42.6% of its maximum activity at 0 °C. rPsPrx was a salt-tolerance enzyme that showed 42.2% of its maximum activity under 2.5 M NaCl. The kinetic parameters of different substrates revealed that it could efficiently catalyze the peroxides, especially H2O2 and t-BOOH (tert­butyl hydroperoxide). Moreover, rPsPrx exhibited the ability to protect super-coiled DNA from oxidative damage. These results indicated that rPsPrx has special catalytic properties and may be a promising candidate for food and industrial applications.

Linear Eyring Plots Conceal a Change in the Rate-Limiting Step in an Enzyme Reaction.

The temperature dependence of psychrophilic and mesophilic ( R)-3-hydroxybutyrate dehydrogenase steady-state rates yields nonlinear and linear Eyring plots, respectively. Solvent viscosity effects and multiple- and single-turnover pre-steady-state kinetics demonstrate that while product release is rate-limiting at high temperatures for the psychrophilic enzyme, either interconversion between enzyme-substrate and enzyme-product complexes or a step prior to it limits the rate at low temperatures. Unexpectedly, a similar change in the rate-limiting step is observed with the mesophilic enzyme, where a step prior to chemistry becomes rate-limiting at low temperatures. This observation may have implications for past and future interpretations of temperature-rate profiles.

Diaminopelargonic acid transaminase from Psychrobacter cryohalolentis is active towards (S)-(-)-1-phenylethylamine, aldehydes and α-diketones.

Substrate and reaction promiscuity is a remarkable property of some enzymes and facilitates the adaptation to new metabolic demands in the evolutionary process. Substrate promiscuity is also a basis for protein engineering for biocatalysis. However, molecular principles of enzyme promiscuity are not well understood. Even for the widely studied PLP-dependent transaminases of class III, the reliable prediction of the biocatalytically important amine transaminase activity is still difficult if the desired activity is unrelated to the natural activity. Here, we show that 7,8-diaminopelargonic acid transaminase (synthase), previously considered to be highly specific, is able to convert (S)-(-)-1-phenylethylamine and a number of aldehydes and diketones. We were able to characterize the (S)-amine transaminase activity of 7,8-diaminopelargonic acid transaminase from Psychrobacter cryohalolentis (Pcryo361) and analyzed the three-dimensional structure of the enzyme. New substrate specificity for α-diketones was observed, though only a weak activity towards pyruvate was found. We examined the organization of the active site and binding modes of S-adenosyl-L-methionine and (S)-(-)-1-phenylethylamine using X-ray analysis and molecular docking. We suggest that the Pcryo361 affinity towards (S)-(-)-1-phenylethylamine arises from the recognition of the hydrophobic parts of the specific substrates, S-adenosyl-L-methionine and 7-keto-8-aminopelargonic acid, and from the flexibility of the active site. Our results support the observation that the conversion of amines is a promiscuous activity of many transaminases of class III and is independent from their natural function. The analysis of amine transaminase activity from among various transaminases will help to make the sequence-function prediction for biocatalysis more reliable.

Allosteric Activation Shifts the Rate-Limiting Step in a Short-Form ATP Phosphoribosyltransferase.

Short-form ATP phosphoribosyltransferase (ATPPRT) is a hetero-octameric allosteric enzyme comprising four catalytic subunits (HisGS) and four regulatory subunits (HisZ). ATPPRT catalyzes the Mg2+-dependent condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N1-(5-phospho-ß-d-ribosyl)-ATP (PRATP) and pyrophosphate, the first reaction of histidine biosynthesis. While HisGS is catalytically active on its own, its activity is allosterically enhanced by HisZ in the absence of histidine. In the presence of histidine, HisZ mediates allosteric inhibition of ATPPRT. Here, initial velocity patterns, isothermal titration calorimetry, and differential scanning fluorimetry establish a distinct kinetic mechanism for ATPPRT where PRPP is the first substrate to bind. AMP is an inhibitor of HisGS, but steady-state kinetics and 31P NMR spectroscopy demonstrate that ADP is an alternative substrate. Replacement of Mg2+ by Mn2+ enhances catalysis by HisGS but not by the holoenzyme, suggesting different rate-limiting steps for nonactivated and activated enzyme forms. Density functional theory calculations posit an SN2-like transition state stabilized by two equivalents of the metal ion. Natural bond orbital charge analysis points to Mn2+ increasing HisGS reaction rate via more efficient charge stabilization at the transition state. High solvent viscosity increases HisGS's catalytic rate, but decreases the hetero-octamer's, indicating that chemistry and product release are rate-limiting for HisGS and ATPPRT, respectively. This is confirmed by pre-steady-state kinetics, with a burst in product formation observed with the hetero-octamer but not with HisGS. These results are consistent with an activation mechanism whereby HisZ binding leads to a more active conformation of HisGS, accelerating chemistry beyond the product release rate.

Biochemical characterization of ParI, an orphan C5-DNA methyltransferase from Psychrobacter arcticus 273-4.

Cytosine-specific DNA methyltransferases are important enzymes in most living organisms. In prokaryotes, most DNA methyltransferases are members of the type II restriction-modification system where they methylate host DNA, thereby protecting it from digestion by the accompanying restriction endonucleases. DNA methyltransferases can also act as solitary enzymes having important roles in controlling gene expression, DNA replication, cell cycle and DNA post-replicative mismatch repair. They have potential applications in biotechnology, such as in labeling of biopolymers, DNA mapping or epigenetic analysis, as well as for general DNA-protein interaction studies. The parI gene from the psychrophilic bacterium Psychrobacter arcticus 273-4 encodes a cytosine-specific DNA methyltransferase. In this work, recombinant ParI was expressed and purified in fusion to either an N-terminal hexahistidine affinity tag, or a maltose binding protein following the hexahistidine affinity tag, for solubility improvement. After removal of the fusion partners, recombinant ParI was found to be monomeric by size exclusion chromatography, with its molecular mass estimated to be 54 kDa. The apparent melting temperature of the protein was 53 °C with no detectable secondary structures above 65 °C. Both recombinant and native ParI showed methyltransferase activity in vivo. In addition, MBP- and His-tagged ParI also demonstrated in vitro activity. Although the overall structure of ParI exhibits high thermal stability, the loss of in vitro activity upon removal of solubility tags or purification from the cellular milieu indicates that the catalytically active form is more labile. Horizontal gene transfer may explain the acquisition of a protein-encoding gene that does not display common cold-adapted features.

Purification and characterization of a novel organic solvent-tolerant and cold-adapted lipase from Psychrobacter sp. ZY124.

By screening 25 different psychrophilic strains isolated from the Arctic habitat, we isolated a strain capable of producing lipase. We identified this strain as Psychrobacter sp. ZY124 based on the amplified 16S rDNA sequence. The lipase, named as Lipase ZC12, produced from the supernatant of Psychrobacter sp. ZY124 cultured at 15 °C was purified to homogeneity by ammonium sulfate precipitation followed by Phenyl Sepharose FF gel hydrophobic chromatography. Based on the obtained amino acid sequence, Lipase ZC12 is classified as a member of the Proteus/psychrophilic subfamily of lipase family I.1; it has a molecular weight of 37.9 kDa. We also determined that the apparent optimum temperature for Lipase ZC12 activity is 40 °C. Lipase ZC12 shows remarkable organic solvent tolerance by remaining more 50% after incubated with 10-90% different organic solvents. In addition, acyl chain esters with C12 or longer were confirmed to be preferable substrates for Lipase ZC12. Lipase ZC12 also shows better stereoselectivity for (R, S)-1-phenylethanol chiral resolution in n-hexane solvent with (S)-1-phenylethanol (eep 92%) and conversion rate (39%) by transesterification reactions. These properties may provide potential applications in biocatalysis and biotransformation in non-aqueous media, such as in detergent, transesterification or esterification and chiral resolution.

One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase.

Enzymes active at low temperature are of great interest for industrial bioprocesses due to their high efficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes is their increased enzymatic activity at low temperature, however the low thermostability presented in this type of enzymes is still a major drawback for their application in biocatalysis. Directed evolution of cold-adapted enzymes to a more thermostable version, appears as an attractive strategy to fulfill the stability and activity requirements for the industry. This paper describes the recombinant expression and characterization of a new and highly active cold-adapted xylanase from the GH-family 10 (Xyl-L), and the use of a novel one step combined directed evolution technique that comprises saturation mutagenesis and focused epPCR as a feasible semi-rational strategy to improve the thermostability. The Xyl-L enzyme was cloned from a marine-Antarctic bacterium, Psychrobacter sp. strain 2-17, recombinantly expressed in E. coli strain BL21(DE3) and characterized enzymatically. Molecular dynamic simulations using a homology model of the catalytic domain of Xyl-L were performed to detect flexible regions and residues, which are considered to be the possible structural elements that define the thermolability of this enzyme. Mutagenic libraries were designed in order to stabilize the protein introducing mutations in some of the flexible regions and residues identified. Twelve positive mutant clones were found to improve the T5015 value of the enzyme, in some cases without affecting the activity at 25°C. The best mutant showed a 4.3°C increase in its T5015. The efficiency of the directed evolution approach can also be expected to work in the protein engineering of stereoselectivity.

Kinetics and Structure of a Cold-Adapted Hetero-Octameric ATP Phosphoribosyltransferase.

Adenosine 5'-triphosphate phosphoribosyltransferase (ATPPRT) catalyzes the first step in histidine biosynthesis, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate to generate N1-(5-phospho-ß-d-ribosyl)-ATP and inorganic pyrophosphate. The enzyme is allosterically inhibited by histidine. Two forms of ATPPRT, encoded by the hisG gene, exist in nature, depending on the species. The long form, HisGL, is a single polypeptide chain with catalytic and regulatory domains. The short form, HisGS, lacks a regulatory domain and cannot bind histidine. HisGS instead is found in complex with a regulatory protein, HisZ, constituting the ATPPRT holoenzyme. HisZ triggers HisGS catalytic activity while rendering it sensitive to allosteric inhibition by histidine. Until recently, HisGS was thought to be catalytically inactive without HisZ. Here, recombinant HisGS and HisZ from the psychrophilic bacterium Psychrobacter arcticus were independently overexpressed and purified. The crystal structure of P. arcticus ATPPRT was determined at 2.34 Å resolution, revealing an equimolar HisGS-HisZ hetero-octamer. Steady-state kinetics indicate that both the ATPPRT holoenzyme and HisGS are catalytically active. Surprisingly, HisZ confers only a modest 2-4-fold increase in kcat. Reaction profiles for both enzymes cannot be distinguished by 31P nuclear magnetic resonance, indicating that the same reaction is catalyzed. The temperature dependence of kcat shows deviation from Arrhenius behavior at 308 K with the holoenzyme. Interestingly, such deviation is detected only at 313 K with HisGS. Thermal denaturation by CD spectroscopy resulted in Tm's of 312 and 316 K for HisZ and HisGS, respectively, suggesting that HisZ renders the ATPPRT complex more thermolabile. This is the first characterization of a psychrophilic ATPPRT.

Isolation and genome sequencing of four Arctic marine Psychrobacter strains exhibiting multicopper oxidase activity.

BACKGROUND: Marine cold-temperature environments are an invaluable source of psychrophilic microbial life for new biodiscoveries. An Arctic marine bacterial strain collection was established consisting of 1448 individual isolates originating from biota, water and sediment samples taken at a various depth in the Barents Sea, North of mainland Norway, with an all year round seawater temperature of 4 °C. The entire collection was subjected to high-throughput screening for detection of extracellular laccase activity with guaiacol as a substrate. RESULTS: In total, 13 laccase-positive isolates were identified, all belonging to the Psychrobacter genus. From the most diverse four strains, based on 16S rRNA gene sequence analysis, all originating from the same Botryllus sp. colonial ascidian tunicate sample, genomic DNA was isolated and genome sequenced using a combined approach of whole genome shotgun and 8 kb mate-pair library sequencing on an Illumina MiSeq platform. The genomes were assembled and revealed genome sizes between 3.29 and 3.52 Mbp with an average G + C content of around 42%, with one to seven plasmids present in the four strains. Bioinformatics based genome mining was performed to describe the metabolic potential of these four strains and to identify gene candidates potentially responsible for the observed laccase-positive phenotype. Up to two different laccase-like multicopper oxidase (LMCO) encoding gene candidates were identified in each of the four strains. Heterologous expression of P11F6-LMCO and P11G5-LMCO2 in Escherichia coli BL21 (DE3) resulted in recombinant proteins exhibiting 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and guaiacol oxidizing activity. CONCLUSIONS: Thirteen Psychrobacter species with laccase-positive phenotype were isolated from a collection of Arctic marine bacteria. Four of the isolates were genome sequenced. The overall genome features were similar to other publicly available Psychrobacter genome sequences except for P11G5 harboring seven plasmids. However, there were differences at the pathway level as genes associated with degradation of phenolic compounds, nicotine, phenylalanine, styrene, ethylbenzene, and ethanolamine were detected only in the Psychrobacter strains reported in this study while they were absent among the other publicly available Psychrobacter genomes. In addition, six gene candidates were identified by genome mining and shown to possess T1, T2 and T3 copper binding sites as the main signature of the three-domain laccases. P11F6-LMCO and P11G5-LMCO2 were recombinantly expressed and shown to be active when ABTS and guaiacol were used as substrates.

A cold-adapted, solvent and salt tolerant esterase from marine bacterium Psychrobacter pacificensis.

Lipolytic enzymes with unique physico-chemical characteristics are gaining more attention for their immense industrial importance. In this study, a novel lipolytic enzyme (Est11) was cloned from the genomic library of a marine bacterium Psychrobacter pacificensis. The enzyme was expressed in Escherichia coli and purified to homogeneity with molecular mass of 32.9kDa. The recombinant Est11 was able to hydrolyze short chain esters (C2-C8) and displayed an optimum activity against butyrate ester (C4). The optimal temperature and pH were 25°C and 7.5, respectively. Est11 retained more than 70% of its original activity at 10°C, suggesting that it was a cold-active esterase. The enzyme was highly active and stable at high concentration of NaCl (5M). Further, incubation with ethanol, isopropanol, propanediol, DMSO, acetonitrile, and glycerol rendered remarkable positive effects on Est11 activity. Typically, even at the concentration of 30% (v/v), ethanol, DMSO, and propanediol increased Est11 activity by 1.3, 2.0, and 2.4-folds, respectively. This new robust enzyme with remarkable properties like cold-adaptability, exceptional tolerance to salt and organic solvents provides us a promising candidate to meet the needs of some harsh industrial processes.

Crystal structure and enzymatic properties of a broad substrate-specificity psychrophilic aminotransferase from the Antarctic soil bacterium Psychrobacter sp. B6.

Aminotransferases (ATs) are enzymes that are commonly used in the chemical and pharmaceutical industries for the synthesis of natural and non-natural amino acids by transamination reactions. Currently, the easily accessible enzymes from mesophilic organisms are most commonly used; however, for economical and ecological reasons the utilization of aminotransferases from psychrophiles would be more advantageous, as their optimum reaction temperature is usually significantly lower than for the mesophilic ATs. Here, gene isolation, protein expression, purification, enzymatic properties and structural studies are reported for the cold-active aromatic amino-acid aminotransferase (PsyArAT) from Psychrobacter sp. B6, a psychrotrophic, Gram-negative strain from Antarctic soil. Preliminary computational analysis indicated dual functionality of the enzyme through the ability to utilize both aromatic amino acids and aspartate as substrates. This postulation was confirmed by enzymatic activity tests, which showed that it belonged to the class EC 2.6.1.57. The first crystal structures of a psychrophilic aromatic amino-acid aminotransferase have been determined at resolutions of 2.19 Šfor the native enzyme (PsyArAT) and 2.76 Šfor its complex with aspartic acid (PsyArAT/D). Both types of crystals grew in the monoclinic space group P21 under slightly different crystallization conditions. The PsyArAT crystals contained a dimer (90 kDa) in the asymmetric unit, which corresponds to the active form of this enzyme, whereas the crystals of the PsyArAT/D complex included four dimers showing different stages of the transamination reaction.

Cell surface display of cold-active esterase EstPc with the use of a new autotransporter from Psychrobacter cryohalolentis K5(T).

We have cloned the gene coding for AT877-a new predicted member of the autotransporter protein family with an esterase passenger domain from permafrost bacterium Psychrobacter cryohalolentis K5(T). Expression of AT877 gene in Escherichia coli resulted in accumulation of the recombinant autotransporter in the outer membrane fraction and at the surface of the induced cells. AT877 displayed maximum hydrolytic activity toward medium-chain p-nitrophenyl esters (C8-C10) at 50 °C and was resistant to the presence of several metal ions, organic solvents and detergents. Previously, we have described a cold-active esterase EstPc from the same bacterium which possesses high activity at low temperatures and relatively high thermal stability. To construct a cell surface display system for EstPc, the hybrid autotransporter gene coding for EstPc with the α-helical linker and the translocator domain from AT877 was constructed and expressed in E. coli. According to the results of the cell fractionation studies and esterase activity measurements, the EstPc passenger was successfully displayed at the surface of the induced cells. It demonstrated a temperature optimum at 15-25 °C and a substrate preference toward p-nitrophenyl butyrate (C4). Obtained results provide a new example of the biotechnologically relevant enzyme from the permafrost microbial community with potential applications for the conversion of short- and medium-chain ester substrates and a basis for the construction of a new cell surface display platform.