Structure and Mechanism of the Ketosynthase-Chain Length Factor Didomain from a Prototypical Polyunsaturated Fatty Acid Synthase.

Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential ingredients of the human diet. They are synthesized by LC-PUFA synthases (PFASs) expressed in marine bacteria and other organisms. PFASs are large enzyme complexes that are homologous to mammalian fatty acid synthases and microbial polyketide synthases. One subunit of each PFAS harbors consecutive ketosynthase (KSc) and chain length factor (CLF) domains that collectively catalyze the elongation of a nascent fatty acyl chain via iterative carbon-carbon bond formation. We report the X-ray crystal structure of the KS-CLF didomain from a well-studied PFAS in Moritella marina. Our structure, in combination with biochemical analysis, provides a foundation for understanding the mechanism of substrate recognition and chain length control by the KS-CLF didomain as well as its interaction with a cognate acyl carrier protein partner.

Effects of Pressure and Temperature on the Atomic Fluctuations of Dihydrofolate Reductase from a Psychropiezophile and a Mesophile.

Determining the effects of extreme conditions on proteins from "extremophilic" and mesophilic microbes is important for understanding how life adapts to living at extremes as well as how extreme conditions can be used for sterilization and food preservation. Previous molecular dynamics simulations of dihydrofolate reductase (DHFR) from a psychropiezophile (cold- and pressure-loving), Moritella profunda (Mp), and a mesophile, Escherichia coli (Ec), at various pressures and temperatures indicate that atomic fluctuations, which are important for enzyme function, increase with both temperature and pressure. Here, the factors that cause increases in atomic fluctuations in the simulations are examined. The fluctuations increase with temperature not only because of greater thermal energy and thermal expansion of the protein but also because hydrogen bonds between protein atoms are weakened. However, the increase in fluctuations with pressure cannot be due to thermal energy, which remains constant, nor the compressive effects of pressure, but instead, the hydrogen bonds are also weakened. In addition, increased temperature causes larger increases in fluctuations of the loop regions of MpDHFR than EcDHFR, and increased pressure causes both increases and decreases in fluctuations of the loops, which differ between the two.

Function of three RuBisCO enzymes under different CO2 conditions in Hydrogenovibrio marinus.

The obligate chemolithoautotrophic bacterium, Hydrogenovibrio marinus MH-110 has three ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) isoenzymes, designated CbbLS-1, CbbLS-2, and CbbM, which are encoded by the cbbL1S1, cbbL2S2, and cbbM genes, respectively. Functions of these isoenzymes at different CO2 concentrations were investigated using deletion mutants of their genes. Deletion of cbbL1 had no effect on cell growth under any of the test growth conditions. The cbbL2 mutant was unable to grow under lower (≤0.15%) CO2 conditions, though it grew normally under higher (≥2%) CO2 conditions. Growth of the cbbM mutant was retarded under higher CO2 conditions but was not affected by lower CO2 conditions. These results indicate that CbbLS-2 and CbbM specifically function under lower and higher CO2 conditions, respectively. The growth retardation of the cbbL2 and cbbM mutants was not restored by complementation with plasmids carrying the cbbL2S2 and cbbM genes, respectively. The cbbL2S2 and cbbM genes are followed by the carboxysome genes and the cbbQmOm genes, respectively. Co-expression of these downstream genes was probably necessary for the in vivo function of CbbLS-2 and CbbM. CbbLS-1 was upregulated in the cbbL2 and cbbM mutants under the lower and higher CO2 conditions, respectively, indicating that the expression of cbbL1S1 was controlled to compensate the deficiency of the other RuBisCO isoenzymes.

Quasiharmonic Analysis of the Energy Landscapes of Dihydrofolate Reductase from Piezophiles and Mesophiles.

A quasiharmonic analysis (QHA) method is used to compare the potential energy landscapes of dihydrofolate reductase (DHFR) from a piezophile (pressure-loving organism), Moritella profunda (Mp), and a mesophile, Escherichia coli (Ec). The QHA method considers atomic fluctuations of the protein as motions of an atom in a local effective potential created by neighboring atoms and quantitates it in terms of effective force constants, isothermal compressibilities, and thermal expansivities. The analysis indicates that the underlying potential energy surface of MpDHFR is inherently softer than that of EcDHFR. In addition, on picosecond time scales, the energy surfaces become more similar under the growth conditions of Mp and Ec. On these time scales, DHFR behaves as expected; namely, increasing temperature makes the effective energy minimum less steep because thermal fluctuations increase the available volume, whereas increasing pressure steepens it because compression reduces the available volume. Our longer simulations show that, on nanosecond time scales, increasing temperature has a similar effect as on picosecond time scales because thermal fluctuations increase the volume even more on a longer time scale. However, these simulations also indicate that, on nanosecond time scales, pressure makes the local potential less steep, contrary to picosecond time scales. Further examination of the QHA indicates the nanosecond pressure response may originate at picosecond time scales at the exterior of the protein, which suggests that protein-water interactions may be involved. The results may lead to understanding adaptations in enzymes made by piezophiles that enable them to function at higher pressures.

Draft Genome Sequence of the Deep-Sea Bacterium Moritella sp. JT01 and Identification of Biotechnologically Relevant Genes.

Deep-sea bacteria can produce various biotechnologically relevant enzymes due to their adaptations to high pressures and low temperatures. To identify such enzymes, we have sequenced the genome of the polycaprolactone-degrading bacterium Moritella sp. JT01, isolated from sediment samples from Japan Trench (6957 m depth), using a Illumina HiSeq2000 sequencer (12.1 million paired-end reads) and CLC Genomics Workbench (version 6.5.1) for the assembly, resulting in a 4.83-Mb genome (42 scaffolds). The genome was annotated using Rapid Annotation using Subsystem Technology (RAST), Protein Homology/analogY Recognition Engine V 2.0 (PHYRE2), and BLAST2Go, revealing 4439 protein coding sequences and 101 RNAs. Gene products with industrial relevance, such as lipases (three) and esterases (four), were identified and are related to bacterium's ability to degrade polycaprolactone. The annotation revealed proteins related to deep-sea survival, such as cold-shock proteins (six) and desaturases (three). The presence of secondary metabolite biosynthetic gene clusters suggests that this bacterium could produce nonribosomal peptides, polyunsaturated fatty acids, and bacteriocins. To demonstrate the potential of this genome, a lipase was cloned an introduced into Escherichia coli. The lipase was purified and characterized, showing activity over a wide temperature range (over 50% at 20-60 °C) and pH range (over 80% at pH 6.3 to 9). This enzyme has tolerance to the surfactant action of sodium dodecyl sulfate and shows 30% increased activity when subjected to a working pressure of 200 MPa. The genomic characterization of Moritella sp. JT01 reveals traits associated with survival in the deep-sea and their potential uses in biotechnology, as exemplified by the characterized lipase.

Cold Adaptation of Triosephosphate Isomerase.

The main problem for enzymes from psychrophilic species, which need to work near the freezing point of liquid water, is the exponential decay of reaction rates as the temperature is decreased. Cold-adapted enzymes have solved this problem by shifting the activation enthalpy-entropy balance for the catalyzed reaction compared to those of their mesophilic orthologs. To understand the structural basis of this universal feature, it is necessary to examine pairs of such orthologous enzymes, with known three-dimensional structures, at the microscopic level. Here, we use molecular dynamics free energy calculations in combination with the empirical valence bond method to evaluate the temperature dependence of the activation free energy for differently adapted triosephosphate isomerases. The results show that the enzyme from the psychrophilic bacterium Vibrio marinus indeed displays the characteristic shift in enthalpy-entropy balance, compared to that of the yeast ortholog. The origin of this effect is found to be located in a few surface-exposed protein loops that show differential mobilities in the two enzymes. Key mutations render these loops more mobile in the cold-adapted triosephosphate isomerase, which explains both the reduced activation enthalpy contribution from the protein surface and the lower thermostability.

Extreme biophysics: Enzymes under pressure.

A critical question about piezophilic (pressure-loving) microbes is how their constituent molecules maintain function under high pressure. Here, factors are examined that may lead to the increased activity under pressure in dihydrofolate reductase from the piezophilic Moritella profunda compared to the homologous enzyme from the mesophilic Escherichia coli. Molecular dynamics simulations are performed at various temperatures and pressures to examine how pressure affects the flexibility of the enzymes from these two microbes, since both stability and flexibility are necessary for enzyme activity. The results suggest that collective motions on the 10-ns timescale are responsible for the flexibility necessary for "corresponding states" activity at the growth conditions of the parent organism. In addition, the results suggest that while the lower stability of many enzymes from deep-sea microbes may be an adaptation for greater flexibility at low temperatures, high pressure may enhance their adaptation to low temperatures. © 2017 Wiley Periodicals, Inc.

Characterization of the N-acetylneuraminic acid synthase (NeuB) from the psychrophilic fish pathogen Moritella viscosa.

Moritella viscosa is a Gram-negative psychrophilic bacterium that causes winter ulcer disease in Atlantic salmon and cod. Its genome reveals that it possesses the ability to synthesize sialic acids. Indeed, sialic acid can be isolated from the bacterium and when analyzed using HPLC-MS/MS, the presence of N-acetylneuraminic acid was confirmed. Thus, the N-acetylneuraminic acid synthase NeuB from M. viscosa (MvNeuB) was recombinantly produced and characterized. The optimum pH and temperature for MvNeuB activity are 7.5 and 30 °C, respectively. The KM for N-acetylmannosamine and phosphoenolpyruvate is 18±5 and 0.8±0.2 mM, respectively. The kcat value (∼225 min(-1)) for both N-acetylmannosamine and phosphoenolpyruvate is the highest turnover number found for an enzyme in this class until the date. A calorimetric study of MvNeuB shows that the enzyme has a two-step transition peak probably reflecting the two domains these proteins consist of. MvNeuB is less stable at higher temperature and has a high catalytic activity at lower temperature compared to mesophilic counterparts. Enzymes from psychrophilic organisms are generally cold adapted meaning they can maintain adequate function near the freezing point of water. Cold adapted enzymes are catalytically more efficient at lower temperature and are more thermo-labile compared to their mesophilic counterparts. MvNeuB is a typical cold adapted enzyme and could be further explored for production of sialic acids and derivates at low temperatures.

Loop interactions during catalysis by dihydrofolate reductase from Moritella profunda.

Dihydrofolate reductase (DHFR) is often used as a model system to study the relation between protein dynamics and catalysis. We have studied a number of variants of the cold-adapted DHFR from Moritella profunda (MpDHFR), in which the catalytically important M20 and FG loops have been altered, and present a comparison with the corresponding variants of the well-studied DHFR from Escherichia coli (EcDHFR). Mutations in the M20 loop do not affect the actual chemical step of transfer of hydride from reduced nicotinamide adenine dinucleotide phosphate to the substrate 7,8-dihydrofolate in the catalytic cycle in either enzyme; they affect the steady state turnover rate in EcDHFR but not in MpDHFR. Mutations in the FG loop also have different effects on catalysis by the two DHFRs. Despite the two enzymes most likely sharing a common catalytic cycle at pH 7, motions of these loops, known to be important for progression through the catalytic cycle in EcDHFR, appear not to play a significant role in MpDHFR.

Role of the occluded conformation in bacterial dihydrofolate reductases.

Dihydrofolate reductase (DHFR) from Escherichia coli (EcDHFR) adopts two major conformations, closed and occluded, and movement between these two conformations is important for progression through the catalytic cycle. DHFR from the cold-adapted organism Moritella profunda (MpDHFR) on the other hand is unable to form the two hydrogen bonds that stabilize the occluded conformation in EcDHFR and so remains in a closed conformation during catalysis. EcDHFR-S148P and MpDHFR-P150S were examined to explore the influence of the occluded conformation on catalysis by DHFR. Destabilization of the occluded conformation did not affect hydride transfer but altered the affinity for the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP(+)) and changed the rate-determining step of the catalytic cycle for EcDHFR-S148P. Even in the absence of an occluded conformation, MpDHFR follows a kinetic pathway similar to that of EcDHFR with product release being the rate-limiting step in the steady state at pH 7, suggesting that MpDHFR uses a different strategy to modify its affinity for NADP(+). DHFRs from many organisms lack a hydrogen bond donor in the appropriate position and hence most likely do not form an occluded conformation. The link between conformational cycling between closed and occluded forms and progression through the catalytic cycle is specific to EcDHFR and not a general characteristic of prokaryotic DHFR catalysis.

Crystal structures of substrate-bound chitinase from the psychrophilic bacterium Moritella marina and its structure in solution.

The four-domain structure of chitinase 60 from Moritella marina (MmChi60) is outstanding in its complexity. Many glycoside hydrolases, such as chitinases and cellulases, have multi-domain structures, but only a few have been solved. The flexibility of the hinge regions between the domains apparently makes these proteins difficult to crystallize. The analysis of an active-site mutant of MmChi60 in an unliganded form and in complex with the substrates NAG4 and NAG5 revealed significant differences in the substrate-binding site compared with the previously determined complexes of most studied chitinases. A SAXS experiment demonstrated that in addition to the elongated state found in the crystal, the protein can adapt other conformations in solution ranging from fully extended to compact.

Structure of a complete four-domain chitinase from Moritella marina, a marine psychrophilic bacterium.

X-ray crystallography reveals chitinase from the psychrophilic bacterium Moritella marina to be an elongated molecule which in addition to the catalytic ß/α-barrel domain contains two Ig-like domains and a chitin-binding domain, all linked in a chain. A ligand-binding study using NAG oligomers showed the enzyme to be active in the crystal lattice and resulted in complexes of the protein with oxazolinium ion (the reaction intermediate) and with NAG2, a reaction product. The characteristic motif DXDXE, containing three acidic amino-acid residues, which is a signature of type 18 chitinases, is conserved in the enzyme. Further analysis of the unliganded enzyme with the two protein-ligand complexes and a comparison with other known chitinases elucidated the roles of other conserved residues near the active site. Several features have been identified that are probably important for the reaction mechanism, substrate binding and the efficiency of the enzyme at low temperatures. The chitin-binding domain and the tryptophan patch on the catalytic domain provide general affinity for chitin, in addition to the affinity of the binding site; the two Ig-like domains give the protein a long reach over the chitin surface, and the flexible region between the chitin-binding domain and the adjacent Ig-like domain suggests an ability of the enzyme to probe the surface of the substrate, while the open shallow substrate-binding groove allows easy access to the active site.

CYP261 enzymes from deep sea bacteria: a clue to conformational heterogeneity in cytochromes P450.

We have explored the adaptation of the cytochromes P450 (P450) of deep-sea bacteria to high hydrostatic pressures. Strict conservation of the protein fold and functional importance of protein-bound water make P450 a unique subject for the studies of high-pressure adaptation. Earlier, we expressed and purified a fatty-acid binding P450 from the deep-sea bacteria Photobacterium profundum SS9 (CYP261C1). Here, we report purification and initial characterization of its mesophilic ortholog from the shallow-water P. profundum 3TCK (CYP261C2), as well as another piezophilic enzyme, CYP261D1, from deep-sea Moritella sp. PE36. Comparison of the three enzymes revealed a striking peculiarity of the piezophilic enzymes. Both CYP261C1 and CYP261D1 possess an apparent pressure-induced conformational toggle actuated at the pressures commensurate with the physiological pressure of habitation of the host bacteria. Furthermore, in contrast to CYP261C2, the piezophilic CYP261 enzymes may be chromatographically separated into two fractions with different properties, and different thermodynamic parameters of spin equilibrium in particular. According to our concept, the changes in the energy landscape that evolved in pressure-tolerant enzymes must stabilize the less-hydrated, closed conformers, which may be transient in the catalytic mechanisms of nonpiezophilic enzymes. The studies of enzymes of piezophiles should help unravel the mechanisms that control water access during the catalytic cycle.

Optimization of cold-active lipase production from psychrophilic bacterium Moritella sp. 2-5-10-1 by statistical experimental methods.

Statistical experimental designs were applied to optimize cold-active lipase production by the psychrophilic bacterium Moritella sp. 2-5-10-1. First, a Plackett-Burmen design (PBD) was used to evaluate the significant effects of various fermentation parameters. The results indicated that soybean meal, temperature, and Tween-80 had significant influences on lipase production. The levels of these variables were optimized subsequently using central composite design (CCD). A quadratic regression model of cold-active lipase production was built, and verification experiments confirmed its validity. On subsequent scale-up in a 10-L bioreactor using optimized conditions, cold-active lipase production (30.56 U/mL) was obtained. The results clearly indicated that the model was adequate even on a large scale. To our knowledge, this is the first report of statistical optimization of cold-active lipase production by a psychrophilic bacterium.

Aliphatic (1)H, (13)C and (15)N chemical shift assignments of dihydrofolate reductase from the psychropiezophile Moritella profunda in complex with NADP(+) and folate.

Dihydrofolate reductase from the deep-sea bacterium Moritella profunda (MpDHFR) has been (13)C/(15)N isotopically labelled and purified. Here, we report the aliphatic (1)H, (13)C and (15)N resonance assignments of MpDHFR in complex with NADP(+) and folate. The spectra of MpDHFR suggest considerably greater conformational heterogeneity than is seen in the closely related DHFR from Escherichia coli.

Pressure dependence of activity and stability of dihydrofolate reductases of the deep-sea bacterium Moritella profunda and Escherichia coli.

To understand the pressure-adaptation mechanism of deep-sea enzymes, we studied the effects of pressure on the enzyme activity and structural stability of dihydrofolate reductase (DHFR) of the deep-sea bacterium Moritella profunda (mpDHFR) in comparison with those of Escherichia coli (ecDHFR). mpDHFR exhibited optimal enzyme activity at 50MPa whereas ecDHFR was monotonically inactivated by pressure, suggesting inherent pressure-adaptation mechanisms in mpDHFR. The secondary structure of apo-mpDHFR was stable up to 80°C, as revealed by circular dichroism spectra. The free energy changes due to pressure and urea unfolding of apo-mpDHFR, determined by fluorescence spectroscopy, were smaller than those of ecDHFR, indicating the unstable structure of mpDHFR against pressure and urea despite the three-dimensional crystal structures of both DHFRs being almost the same. The respective volume changes due to pressure and urea unfolding were -45 and -53ml/mol at 25°C for mpDHFR, which were smaller (less negative) than the corresponding values of -77 and -85ml/mol for ecDHFR. These volume changes can be ascribed to the difference in internal cavity and surface hydration of each DHFR. From these results, we assume that the native structure of mpDHFR is loosely packed and highly hydrated compared with that of ecDHFR in solution.

Reduced susceptibility of Moritella profunda dihydrofolate reductase to trimethoprim is not due to glutamate 28.

The E28D variant of dihydrofolate reductase from Moritella profunda was generated and found to have the same K (i) (within error) for the competitive inhibitor trimethoprim as the wild type enzyme. Contrary to a previous claim in the literature, Glu 28 is therefore not the cause of the reduced affinity for trimethoprim relative to dihydrofolate reductase from Escherichia coli.

Catalysis by dihydrofolate reductase from the psychropiezophile Moritella profunda.

The influence of temperature and pH on the stability and catalytic activity of dihydrofolate reductase (MpDHFR) from the cold-adapted deep-sea bacterium Moritella profunda was studied. The thermal melting temperature was found to be ∼38 °C and was not affected by pH, while activity measurements demonstrated that its stability was maximal at pH 7 and was reduced dramatically below pH 6 or above pH 8. The steady-state rate constant (k(cat)) was maximal at neutral pH and higher temperatures, while the Michaelis constants (K(M)) for both substrate and cofactor were optimal at lower temperatures and at elevated or reduced pH. For both temperature and pH, any change in k(cat) was therefore offset by a similar change in K(M). Both the activation enthalpy and entropy of the MpDHFR-catalysed reaction were lower than those of DHFR from E. coli leading overall to a very small difference in activation free energy and therefore similar steady-state rate constants at the same temperature. The chemical step of the reaction is not rate limiting at pH 7, but becomes progressively more rate limiting as the pH increases. These results demonstrate adaptation of MpDHFR to its environment and show compromises between enthalpic and entropic contributions to the reaction, and between k(cat) and K(M).

Cloning and characterization of dihydrofolate reductases from deep-sea bacteria.

Enzymes from organisms living in deep-sea are thought to have characteristic pressure-adaptation mechanisms in structure and function. To better understand these mechanisms in dihydrofolate reductase (DHFR), an essential enzyme in living cells, we cloned, overexpressed and purified four new DHFRs from the deep-sea bacteria Shewanella violacea (svDHFR), Photobacterium profundum (ppDHFR), Moritella yayanosii (myDHFR) and Moritella japonica (mjDHFR), and compared their structure and function with those of Escherichia coli DHFR (ecDHFR). These deep-sea DHFRs showed 33-56% primary structure identity to ecDHFR while far-ultraviolet circular dichroism and fluorescence spectra suggested that their secondary and tertiary structures were not largely different. The optimal temperature and pH for deep-sea DHFRs activity were lower than those of ecDHFR and different from each other. Deep-sea DHFRs kinetic parameters K(m) and k(cat) were larger than those of ecDHFR, resulting in 1.5-2.8-fold increase of k(cat)/K(m) except for mjDHFR which had a 28-fold decrease. The enzyme activity of ppDHFR and mjDHFR (moderate piezophilic bacteria) as well as ecDHFR decreased as pressure increased, while svDHFR and myDHFR (piezophilic bacteria) showed a significant tolerance to pressure. These results suggest that DHFRs from deep-sea bacteria possess specific enzymatic properties adapted to their life under high pressure.

Are the catalytic properties of enzymes from piezophilic organisms pressure adapted?

We report the crystal structure of dihydrofolate reductase (DHFR) from the psychropiezophilic bacterium Moritella profunda, which was isolated from the deep ocean at 2 degrees C and 280 bar. The structure is typical of a chromosomal DHFR and we were unable to identify any obvious structural features that would suggest pressure adaptation. In particular, the core regions of the enzyme are virtually identical to those of the DHFR from the mesophile Escherichia coli. The steady-state rate at pH 9, which is limited by hydride transfer at atmospheric pressure, is roughly constant between 1 and 750 bar, falling at higher pressures. However, the value of K(M) increases with increasing pressure, and as a result k(cat)/K(M) decreases over the entire pressure range studied. Isotope effect studies showed that increasing the pressure causes a change in the rate-limiting step of the reaction. We therefore see no evidence of pressure adaptation in either the structure or the activity of this enzyme.