Regulation of Shigella Effector Kinase OspG through Modulation of Its Dynamic Properties

Gram-negative pathogens secrete effector proteins into human cells to modulate normal cellular processes and establish a bacterial replication niche. Shigella and pathogenic Escherichia coli possess homologous effector kinases, OspG and NleH1/2, respectively. Upon translocation, OspG but not NleH binds to ubiquitin and a subset of E2 ~ Ub conjugates, which was shown to activate its kinase activity. Here we show that OspG, having a minimal kinase fold, acquired a novel mechanism of regulation of its activity. Binding of the E2 ~ Ub conjugate to OspG not only stimulates its kinase activity but also increases its optimal temperature for activity to match the human body temperature and stabilizes its labile C-terminal domain. The melting temperature (T_m) of OspG alone is only 31?°C, as compared to 41?°C to NleH1/2 homologs. In the presence of E2 ~ Ub, the T_m of OspG increases to ~ 42?°C, while Ub by itself increases the T_m to 39?°C. Moreover, OspG alone displays maximal activity at 26?°C, while in the presence of E2 ~ Ub, maximal activity occurs at ~ 42?°C. Using NMR and molecular dynamics calculations, we have identified the C-terminal lobe and, in particular, the C-terminal helix, as the key elements responsible for lower thermal stability of OspG as compared to homologous effector kinases.     

Construction of recombinant Escherichia coli for enhanced bioconversion of colchicine into 3-demethylated colchicine at 70 l bioreactor level

A recombinant strain of Escherichia coli with CYP102A1 gene was developed for the demethylation of colchicine into their derivatives. The CYP102A1 gene responsible for demethylation was isolated from Bacillus megaterium ACBT03 and amplified using suitable primers. The amplified product was cloned into pET28a+ expression vector using host E. coli BL21(DE3) cells. The CYP3A4 (product of CYP102A1 gene) protein expression and other parameters like substrate toxicity, product toxicity and enzyme activity were optimized in shake flasks; and further scaled-up to 5 l bioreactor with 3 l working volume. In 5 l bioreactor, dissolved oxygen (DO) was optimized for maximum specific growth and enhanced 3-demethylated colchicine (3-DMC) production. The optimized conditions from shake flasks were scaled-up to 70 l bioreactor and resulted into ∼80% conversion of 20 mM colchicine in 48 h with a volumetric productivity of 6.62 mg l⁻¹ h⁻¹. Scale-up factors were measured as volumetric oxygen transfer coefficient (k_La) i.e., 56 h⁻¹ and impeller tip velocity (V_tip) i.e., 7.065 m s⁻¹, respectively. The kinetic parameters K_m, k_cat, and k_cat/K_m of the CYP3A4 enzyme using colchicine as the substrate were determined to be 271 ± 30 μM, 8533 ± 25 min⁻¹, and 31.49 μM min⁻¹, respectively, when IPTG induced recombinant E. coli culture was used.     

Sap flow and water use in African baobab (Adansonia digitata L.) seedlings in response to drought stress

The African baobab (Adansonia digitata L.) is an important multi-purpose fruit tree with high potential for domestication in drier Africa. Although adult individuals are well-known to be drought resistant, only little has been reported on how young baobab trees can survive drought. Therefore, the aim of this study was to examine short-term soil drought effects on water relations of baobab seedlings. Baobab seedlings used a limited amount of stored water to buffer daily water deficits (~ 8.5 g d^− 1), which contributed up to only ~ 17.5% of daily water use and ~ 6% of total plant water. Under drought, a strong reduction in stomatal conductance (~ 85%) resulted in a midday leaf water potential of − 1 MPa and zero stem sap flow followed by significant leaf loss. Plant anatomy evidenced the presence of water storage tissues and the vulnerability to xylem embolism. The taproot was the most important plant part for water storage (68% of total plant water), suggesting root-succulence rather than stem-succulence. When drought intensified, limitation of leaf transpiration and/or root water uptake led to drought-enforced dormancy. Despite the large amounts of water stored in the taproot (~ 90%) and the stem (~ 75%), only a limited amount of stored water appeared to be used to sustain upper leaves and plant metabolism during the dormant period, and to facilitate recovery following water supply. Drought avoidance, conservative water use and the presence of internal stored water allow baobab seedlings to survive drought.     

Identification and evaluation of nutrient limitation on periphyton growth in headwater streams in the Pawnee Nation,Oklahoma

The objective of the study was to identify nutrient impacts, if any, on stream periphyton growth in Black Bear Creek (north central Oklahoma) and its tributaries. Passive diffusion periphytometers were deployed at ten study sites within the Black Bear Creek basin to evaluate periphyton growth in response to nutrient enrichment. These sites were selected to represent a gradient of land uses, from predominantly agricultural to predominantly urban. Periphytometer treatments included phosphorus (P) (1.0 mg/L PO₄-P, n = 10), nitrogen (N) (10.0 mg/L NO₃-N, n = 10), N plus P (n = 10) and control (reverse osmosis-treated water, n = 10). Results indicated that average dissolved inorganic N (DIN, PQL = 0.04 mg/L) concentrations were significantly correlated (R² = 0.63, p < 0.01) with chlorophyll a production on the periphytometer control treatments in the Black Bear Creek basin. Periphytic growth was nutrient-limited (increased chlorophyll a was measured on nutrient-enriched growth media) at four of the ten sites sampled; two sites were limited by N and two sites were co-limited by both N and P. The lotic ecosystem trophic status index (LETSI), the ratio of C to N + P chlorophyll a, was calculated to compare treatment responses across sites. At nutrient-limited sites, LETSI was positively correlated to ambient DIN values (R² = 0.97, p < 0.01). However, some sites that were not nutrient-limited had ambient nutrient concentrations similar to sites with observed nutrient limitation, indicating other factors were limiting periphyton growth at those sites.     

Pure MnTBAP selectively scavenges peroxynitrite over superoxide: Comparison of pure and commercial MnTBAP samples to MnTE-2-PyP in two models of oxidative stress injury,an SOD-specific Escherichia coli model and carrageenan-induced pleurisy

MnTBAP is often referred to as an SOD mimic in numerous models of oxidative stress. We have recently reported that pure MnTBAP does not dismute superoxide, but commercial or poorly purified samples are able to perform O₂^·?dismutation with low-to-moderate efficacy via non-innocent Mn-containing impurities. Herein, we show that neither commercial nor pure MnTBAP could substitute for SOD enzyme in a SOD-deficient Escherichia coli model, whereas MnTE-2-PyP-treated SOD-deficient E. coli grew as well as a wild-type strain. This SOD-specific system indicates that MnTBAP does not act as an SOD mimic in vivo. In another model, carrageenan-induced pleurisy in mice, inflammation was evidenced by increased pleural fluid exudate and neutrophil infiltration and activation: these events were blocked by 0.3 mg/kg MnTE-2-PyP and, to a slightly lesser extent, by 10 mg/kg of either MnTBAP. Also, 3-nitrotyrosine formation, an indication of peroxynitrite existence in vivo, was blocked by both compounds; again MnTE-2-PyP was 33-fold more effective. Pleurisy model data indicate that MnTBAP exerts some protective actions in common with MnTE-2-PyP, which are not O₂^·? related and can be fully rationalized if one considers that the common biological role shared by MnTBAP and MnTE-2-PyP is related to their reduction of peroxynitrite and carbonate radical, the latter arising from ONOOCO₂ adduct. The log k_cat (O₂^·?) value for MnTBAP is estimated to be about 3.16, which is ~ 5 and ~ 6 orders of magnitude smaller than the SOD activities of the potent SOD mimic MnTE-2-PyP and Cu,Zn-SOD, respectively. This very low value indicates that MnTBAP is too inefficient at dismuting superoxide to be of any biological impact, which was confirmed in the SOD-deficient E. coli model. The peroxynitrite scavenging ability of MnTBAP, however, is only ~ 2.5 orders of magnitude smaller than that of MnTE-2-PyP and is not significantly affected by the presence of the SOD-active impurities in the commercial MnTBAP sample (log k_red (ONOO^?) = 5.06 for pure and 4.97 for commercial sample). The reduction of carbonate radical is equally fast with MnTBAP and MnTE-2-PyP. The dose of MnTBAP required to yield oxidative stress protection and block nitrotyrosine formation in the pleurisy model is > 1.5 orders of magnitude higher than that of MnTE-2-PyP, which could be related to the lower ability of MnTBAP to scavenge peroxynitrite. The slightly better protection observed with the commercial MnTBAP sample (relative to the pure MnTBAP) could arise from its impurities, which, by scavenging O₂^·?, reduce consequently the overall peroxynitrite and secondary ROS/RNS levels. These observations have profound biological repercussions as they may suggest that the effect of MnTBAP observed in numerous studies may conceivably relate to peroxynitrite scavenging. Moreover, provided that pure MnTBAP is unable to dismute superoxide at any significant extent, but is able to partially scavenge peroxynitrite and carbonate radical, this compound may prove valuable in distinguishing ONOO^?/CO₃^·? from O₂^·? pathways.     

In vivo temperature limitations of photosynthesis in Phaseolus vulgaris L

The purpose of this study was to evaluate the temperature response of photosynthesis in two common bean genotypes differing in crop yield when grown under warm conditions. The cultivar Nobre is sensitive to high temperatures, whereas Diplomata shows better crop yield under high temperatures. Plants were grown in a greenhouse prior to transferring to a controlled environment cabinet for the temperature treatments. In a first experiment, 30 days-old plants were subjected to a short exposure (1 day) at temperatures that varied from 9 °C to 39 °C. Diplomata had lower net CO₂ assimilation rate (A) at 15 °C and 21 °C, but higher from 27 °C to 39 °C. Photosynthetic parameters calculated from modeling the response of A to the intercellular CO₂ concentration suggested that the different temperature responses of the two genotypes are caused by different rates of diffusion of CO₂ to the assimilation site, not by differences in biochemical limitations of photosynthesis. While stomatal conductance (g_s) did not differ between the genotypes, mesophyll conductance (g_m) was slightly greater for Nobre at 15 °C, but much higher in Diplomata from 21 °C to 39 °C. In a second experiment, no difference was observed in biomass accumulation between the two genotypes after growth for 24 days under a 35/20 °C (day/night) regime. Hence, the differences in photosynthesis did not cause variation in plant growth at the vegetative stage. The differential genotypic response of g_m to temperature suggests that g_m might be an important limitation to photosynthesis in Nobre, the common bean genotype sensitive to elevated temperature. However, more studies are needed employing other methods for g_m evaluation to validate these results.     

Tropical epiphytes in a CO2-rich atmosphere

We tested the effect on epiphyte growth of a doubling of pre-industrial CO₂ concentration (280 vs. 560 ppm) combined with two light (three fold) and two nutrition (ten fold) treatments under close to natural humid conditions in daylight growth cabinets over 6 months. Across co-treatments and six species, elevated CO₂ increased relative growth rates by only 6% (p = 0.03). Although the three C3 species, on average, grew 60% faster than the three CAM species, the two groups did not significantly differ in their CO₂ response. The two Orchidaceae, Bulbophyllum (CAM) and Oncidium (C3) showed no CO₂ response, and three out of four Bromeliaceae showed a positive one: Aechmea (CAM, +32% p = 0.08), Catopsis (C3, +11% p = 0.01) and Vriesea (C3, +4% p = 0.02). In contrast, the representative of the species-rich genus Tillandsia (CAM), which grew very well under experimental conditions, showed no stimulation. On average, high light increased growth by 21% and high nutrients by 10%. Interactions between CO₂, light and nutrient treatments (low vs. high) were inconsistent across species. CO₂ responsive taxa such as Catopsis, could accelerate tropical forest dynamics and increase branch breakage, but overall, the responses to doubling CO₂ of these epiphytes was relatively small and the responses were taxa specific.     

Escherichia coli HGT: Engineered for high glucose throughput even under slowly growing or resting conditions

Aerobic production-scale processes are constrained by the technical limitations of maximum oxygen transfer and heat removal. Consequently, microbial activity is often controlled via limited nutrient feeding to maintain it within technical operability. Here, we present an alternative approach based on a newly engineered Escherichia coli strain. This E. coli HGT (high glucose throughput) strain was engineered by modulating the stringent response regulation program and decreasing the activity of pyruvate dehydrogenase. The strain offers about three-fold higher rates of cell-specific glucose uptake under nitrogen-limitation (0.6 g_Glc g_CDW⁻¹ h⁻¹) compared to that of wild type, with a maximum glucose uptake rate of about 1.8 g_Glc g_CDW⁻¹ h⁻¹ already at a 0.3 h⁻¹ specific growth rate. The surplus of imported glucose is almost completely available via pyruvate and is used to fuel pyruvate and lactate formation. Thus, E. coli HGT represents a novel chassis as a host for pyruvate-derived products.     

Regulation of spiroimine neurotoxins and hemolytic activity in laboratory cultures of the dinoflagellate Alexandrium peruvianum (Balech & Mendiola) Balech & Tangen

Defined experimental regimes were used to determine the effects of nutrient limitation on the toxicity of Alexandrium peruvianum in batch culture. Subsamples for cell counts and spiroimine analysis at six day intervals were used to investigate the concentrations and composition of these compounds throughout growth. An erythrocyte lysis assay for hemolytic activity was performed on cell pellets and supernatants also collected every six days over the entire growth period from all treatments. From the data, growth rates, cellular spiroimine quotas and effective concentration-fifty (EC₅₀s) for cellular and supernatant associated hemolytic activity were calculated. Phosphate limitation was identified as a key regulator of toxicity in this species, yielding maximum values of 54.1 pg cell⁻¹ for 13-desmethyl spirolide C, 96.4 pg cell⁻¹ for 12-methylgymnodimine and a potent hemolytic EC₅₀ value of 7.1 × 10³ cells. The concentrations of spiroimines detected in A. peruvianum among various treatments, in addition to a unique profile of paralytic shellfish poisoning toxins, is unique in the body of microalgal literature. Because of the multiple toxin arsenal produced by this organism, the evaluation of a single toxin clearly would have underestimated the potential virulence and significance of this clone. This study provides the first evidence that growth and toxin production of A. peruvianum are influenced by altered nutrient ratios.     

Effects of nutrients on arsenic accumulation by arsenic hyperaccumulator Pteris vittata L.

This research investigated the effects of various nutrients on arsenic (As) removal by arsenic hyperaccumulator Pteris vittata L. in a Hoagland nutrient solution (HNS). The treatments included different concentrations of Ca and K in 20% strength of HNS, different strengths of HNS (10, 20 and 30%), different strengths of HNS (10 and 20%) with and without CaCO₃, and different concentrations of Ca, K, NO₃, NH₄, and P in 20% strength of HNS. The plants were grown in nutrient solution containing 1 mg As L^?1 for 4 weeks except the Ca/K experiment where the plants were grown in nutrient solution containing 10 or 50 mg As L^?1 for 1 week. Adding up to 4 mM Ca or 3 mM K to 20% strength HNS significantly (P < 0.05) increased plant arsenic accumulation when the solution contained 10 mg As L^?1. Plant arsenic removal was reduced with increasing Ca and K concentrations at 50 mg As L^?1. Lower strength of HNS (10%) resulted in the greatest plant arsenic removal (79%) due to lower competition of P with As for plant uptake. Addition of CaCO₃ to 20% strength of HNS significantly increased arsenic removal by P. vittata. Among the nutrients tested, NO₃ and CaCO₃ were beneficial to plant arsenic removal while NH₄, P and Cl had adverse effects. This experiment demonstrated that it is possible to optimize plant arsenic removal by adjusting nutrients in the growth medium.     

Structural changes that occur upon photolysis of the Fe(II)a3–CO complex in the cytochrome ba3-oxidase of Thermus thermophilus: A combined X-ray crystallographic and infrared spectral study demonstrates CO binding to CuB

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a₃ moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba₃-oxidase from Thermus thermophilus, determined at ~ 2.8–3.2 Å resolution, reveal a Fe–C distance of ~ 2.0 Å, a Cu–O distance of 2.4 Å and a Fe–C–O angle of ~ 126°. Upon photodissociation at 100 K, X-ray structures indicate loss of Fe_a3–CO and appearance of Cu_B–CO having a Cu–C distance of ~ 1.9 Å and an O–Fe distance of ~ 2.3 Å. Absolute FTIR spectra recorded from single crystals of reduced ba₃–CO that had not been exposed to X-ray radiation, showed several peaks around 1975 cm^? 1; after photolysis at 100 K, the absolute FTIR spectra also showed a significant peak at 2050 cm^? 1. Analysis of the ‘light’ minus ‘dark’ difference spectra showed four very sharp CO stretching bands at 1970 cm^? 1, 1977 cm^? 1, 1981 cm^? 1, and 1985 cm^? 1, previously assigned to the Fe_a3–CO complex, and a significantly broader CO stretching band centered at ~ 2050 cm^? 1, previously assigned to the CO stretching frequency of Cu_B bound CO. As expected for light propagating along the tetragonal axis of the P4₃2₁2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba₃ crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO₂ at 2337 cm^? 1 and one from traces of CO at 2133 cm^? 1; while bands associated with CO bound to either Fe_a3 or to Cu_B in “light” minus “dark” FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2 Å and FTIR spectra support the long-held position that photolysis of Fe_a3–CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu_B atom; Fe_a3 and Cu_B ligation, at the resolutions reported here, are otherwise unaltered. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Construction of a non toxic chimeric protein (L1–L2–B) of Haemolysin BL from Bacillus cereus and its application in HBL toxin detection

Among the many potential virulence factors of B. cereus, Haemolysin BL is a unique and potent three component pore forming toxin composed of a binding component, B, and two lytic components, L₁ and L₂. Heterogeneity in nucleic acid and protein sequences of HBL components and problems during expression of L₁ and L₂ proteins in recombinant host due to their toxicity causes problems for development of specific detection systems based on PCR and Immunoassay, respectively. Commercially available kit (BCET RPLA, Oxoid) is useful for detection of L₂ component of HBL, but detection of only one component is insufficient to give comprehensive view on HBL toxin producing strains as some strains produced only one or two of the three HBL components. To address above mentioned problems, in this study, we cloned conserved domains of B, L₁ and L₂ components together as single fusion gene and expressed as recombinant multidomain chimeric protein in E. coli. The resultant protein having L₁, B and L₂ components in the form of single protein had no toxicity towards E. coli as we followed truncated protein approach. The hyperimmune antisera raised in mice against r-chimeric protein reacted with all the three components of HBL toxin of B. cereus (ATCC 14579) and provided three reaction bands at ~ 40 kDa to ~ 50 kDa regions during Western blot analysis. The hyperimmune sera of r-chimeric protein also notably neutralized the hemolytic activity of native HBL toxin. These results demonstrated that the obtained chimeric protein is correct and retained the antigenicity of native HBL toxin components. Therefore, it has better application in the development of a comprehensive HBL detection immunoassay and may also be a potential candidate molecule for vaccine studies.     

Purification,kinetic and thermodynamic characterization of soluble acid invertase from sugarcane (Saccharum officinarum L.)

We report for the first time kinetic and thermodynamic properties of soluble acid invertase (SAI) of sugarcane (Saccharum officinarum L.) salt sensitive local cultivar CP 77-400 (CP-77). The SAI was purified to apparent homogeneity on FPLC system. The crude enzyme was about 13 fold purified and recovery of SAI was 35%. The invertase was monomeric in nature and its native molecular mass on gel filtration and subunit mass on SDS-PAGE was 28 kDa. SAI was highly acidic having an optimum pH lower than 2. The acidic limb was missing. Proton transfer (donation and receiving) during catalysis was controlled by the basic limb having a pKa of 2.4. Carboxyl groups were involved in proton transfer during catalysis. The kinetic constants for sucrose hydrolysis by SAI were determined to be: k_m = 55 mg ml^?1, k_cat = 21 s^?1, k_cat/k_m = 0.38, while the thermodynamic parameters were: ΔH* = 52.6 kJ mol^?1, ΔG* = 71.2 kJ mol^?1, ΔS* = ?57 J mol^?1 K^?1, ΔG*_E–S = 10.8 kJ mol^?1 and ΔG*_E–T = 2.6 kJ mol^?1. The kinetics and thermodynamics of irreversible thermal denaturation at various temperatures 53–63 °C were also determined. The half -life of SAI at 53 and 63 °C was 112 and 10 min, respectively. At 55 °C, surprisingly the half -life increased to twice that at 53 °C. ΔG*, ΔH* and ΔS* of irreversible thermal stability of SAI at 55 °C were 107.7 kJ mol^?1, 276.04 kJ mol^?1 and 513 J mol^?1K^?1, respectively.     

Sulforaphane improves dysregulated metabolic profile and inhibits leptin-induced VSMC proliferation: Implications toward suppression of neointima formation after arterial injury in western diet-fed obese mice

Sulforaphane (SFN), a dietary phase-2 enzyme inducer that mitigates cellular oxidative stress through nuclear factor erythroid 2-related factor 2 (Nrf2) activation, is known to exhibit beneficial effects in the vessel wall. For instance, it inhibits vascular smooth muscle cell (VSMC) proliferation, a major event in atherosclerosis and restenosis after angioplasty. In particular, SFN attenuates the mitogenic and pro-inflammatory actions of platelet-derived growth factor (PDGF) and tumor necrosis factor-α (TNFα), respectively, in VSMCs. Nevertheless, the vasoprotective role of SFN has not been examined in the setting of obesity characterized by hyperleptinemia and insulin resistance. Using the mouse model of western diet-induced obesity, the present study demonstrates for the first time that subcutaneous delivery of SFN (0.5 mg/Kg/day) for ~ 3 weeks significantly attenuates neointima formation in the injured femoral artery [↓ (decrease) neointima/media ratio by ~ 60%; n = 5–8]. This was associated with significant improvements in metabolic parameters, including ↓ weight gain by ~ 52%, ↓ plasma leptin by ~ 42%, ↓ plasma insulin by ~ 63%, insulin resistance [↓ homeostasis model assessment of insulin resistance (HOMA-IR) index by ~ 73%], glucose tolerance (↓ AUC_GTT by ~ 24%), and plasma lipid profile (e.g., ↓ triglycerides). Under in vitro conditions, SFN significantly decreased leptin-induced VSMC proliferation by ~ 23% (n = 5) with associated diminutions in leptin-induced cyclin D1 expression and the phosphorylation of p70S6kinase and ribosomal S6 protein (n = 3–4). The present findings reveal that, in addition to improving systemic metabolic parameters, SFN inhibits leptin-induced VSMC proliferative signaling that may contribute in part to the suppression of injury-induced neointima formation in diet-induced obesity.     

Pseudo-enzymatic hydrolysis of 4-nitrophenyl acetate by human serum albumin: pH-dependence of rates of individual steps

Human serum albumin (HSA) displays esterase activity reflecting multiple irreversible chemical modifications rather than turnover. Here, kinetics of the pseudo-enzymatic hydrolysis of 4-nitrophenyl acetate (NphOAc) are reported. Under conditions where [HSA] ? 5×[NphOAc] and [NphOAc] ? 5×[HSA], the HSA-catalyzed hydrolysis of NphOAc is a first-order process for more than 95% of its course. From the dependence of the apparent rate constants k_app and k_obs on [HSA] and [NphOAc], respectively, values of K_s, k₊₂, and k₊₂/K_s were determined. Values of K_s, k₊₂, and k₊₂/K_s obtained at [HSA] ? 5×[NphOAc] and [NphOAc] ? 5×[HSA] are in good agreement, the deacylation step being rate limiting in catalysis. The pH-dependence of k₊₂/K_s, k₊₂, and K_s reflects the acidic pK_a shift of the Tyr411 catalytic residue from 9.0 ± 0.1 in the substrate-free HSA to 8.1 ± 0.1 in the HSA:NphOAc complex. Accordingly, diazepam inhibits competitively the HSA-catalyzed hydrolysis of NphOAc by binding to Tyr411.     

Comparison of two type IV hyperthermophilic adenylyl cyclases characterizations from the archaeon Pyrococcus furiosus

In this paper, two genes that encoded two soluble type IV adenylyl cyclases (AC) from the hyperthermophilic archaeon Pyrococcus furiosus (PFAC I and PFAC II) were cloned and expressed in Escherichia coli (E. coli) BL21 (DE3). Amino acid sequence analysis of the two enzymes showed 29% homology. PFAC I and PFAC II were both Mn²⁺ activated enzyme. They were purified by His-trap chromatography and had a specific activity of 3.1 × 10³ U/mg at pH 10.0, 95 °C (PFAC I) and 2.0 × 10³ U/mg at pH 11.0, 95 °C (PFAC II), respectively. The K_m and k_cat of PFAC I was 1.38 mM and 1.11 s⁻¹. The K_m and k_cat of PFAC II was 1.44 mM and 0.80 s⁻¹. The thermostability of PFAC I and PFAC II were higher than the soluble type IV adenylyl cyclases from Yersinia pestis (YpAC-IV). All of the properties suggested that these two adenylyl cyclases may be useful for the industrial producing of cyclic adenosine 3′,5′-monophosphate (cAMP).     

Phenoloxidase activity and thermostability of Cancer pagurus and Limulus polyphemus hemocyanin

The intrinsic and inducible o-diphenoloxidase (o-diPO) activity of Cancer pagurus hemocyanin (CpH) and Limulus polyphemus hemocyanin (LpH) were studied using catechol, l-Dopa and dopamine as substrates. The kinetic analysis shows that dopamine is a more specific substrate for CpH than catechol and l-Dopa (K_m value of 0.01 mM for dopamine versus 0.67 mM for catechol, and 2.14 mM for l-Dopa), while k_cat is highest for catechol (2.44 min^? 1 versus 0.67 min^? 1 for l-Dopa and 0.71 min^? 1 for dopamine). On treatment with 4 mM sodium dodecyl sulfate (SDS) or by proteolysis the o-diPO activity of CpH increases about twofold. In contrast, native LpH shows no o-diPO activity, and exhibits only a slight activity after incubation with SDS. Neither CpH nor LpH show intrinsic mono-PO activity with l-tyrosine and tyramine as substrates. To explore the possible correlation between the degree of PO activity and protein stability of arthropod hemocyanins, the thermal stability of CpH and LpH was investigated by differential scanning calorimetry and Fourier transform infrared spectroscopy. CpH is found to be less thermostable (T_m ~ 80 °C), suggesting that the dicopper active sites are more accessible, thereby allowing the hemocyanin to show PO activity in the native state. The LpH, on the other hand, is more thermostable (T_m ~ 92 °C), suggesting the existence of a correlation between the thermal stability and the intrinsic PO activity of arthropod hemocyanins.     

Structural and functional analyses of Mycobacterium tuberculosis Rv3315c-encoded metal-dependent homotetrameric cytidine deaminase

The emergence of drug-resistant strains of Mycobacterium tuberculosis, the causative agent of tuberculosis, has exacerbated the treatment and control of this disease. Cytidine deaminase (CDA) is a pyrimidine salvage pathway enzyme that recycles cytidine and 2′-deoxycytidine for uridine and 2′-deoxyuridine synthesis, respectively. A probable M. tuberculosis CDA-coding sequence (cdd, Rv3315c) was cloned, sequenced, expressed in Escherichia coli BL21(DE3), and purified to homogeneity. Mass spectrometry, N-terminal amino acid sequencing, gel filtration chromatography, and metal analysis of M. tuberculosis CDA (MtCDA) were carried out. These results and multiple sequence alignment demonstrate that MtCDA is a homotetrameric Zn²⁺-dependent metalloenzyme. Steady-state kinetic measurements yielded the following parameters: K_m = 1004 μM and k_cat = 4.8 s^?1 for cytidine, and K_m = 1059 μM and k_cat = 3.5 s^?1 for 2′-deoxycytidine. The pH dependence of k_cat and k_cat/K_M for cytidine indicate that protonation of a single ionizable group with apparent pK_a value of 4.3 abolishes activity, and protonation of a group with pK_a value of 4.7 reduces binding. MtCDA was crystallized and crystal diffracted at 2.0 Å resolution. Analysis of the crystallographic structure indicated the presence of a Zn²⁺ coordinated by three conserved cysteines and the structure exhibits the canonical cytidine deaminase fold.     

pH-rate profiles of l-arabinitol 4-dehydrogenase from Hypocrea jecorina and its application in l-xylulose production

l-Arabinitol 4-dehydrogenase (LAD) from Hypocrea jecorina (HjLAD) was cloned and overexpressed in Escherichia coli BL21 (DE3). The kinetics of l-arabinitol oxidation by NAD⁺, catalyzed by HjLAD, was studied within the pH range of 7.0–9.5 at 25 °C. The turnover number (k_cat) and the catalytic efficiency (k_cat/K_m) were 4200 min⁻¹ and 290 mM⁻¹ min⁻¹, respectively. HjLAD showed the highest turnover number and catalytic efficiency among all previously characterized LADs. In further application of HjLAD, rare l-sugar l-xylulose was produced by the enzymatic oxidation of arabinitol to give a yield of approximately 86%.