Temperature dependence of nitrate reductase in the psychrophilic unicellular alga Koliella antarctica and the mesophilic alga Chlorella sorokiniana

Abstract

Temperature responses of nitrate reductase (NR) were studied in the psychrophilic unicellular alga, Koliella antarctica , and in the mesophilic species, Chlorella sorokiniana . Enzymes from both species were purified to near homogeneity by Blue Sepharose (Pharmacia, Uppsala, Sweden) affinity chromatography and high-resolution anionexchange chromatography (MonoQ; Pharmacia; Uppsala, Sweden). Both enzymes have a subunit molecular mass of 100 kDa, and K. antarctica NR has a native molecular mass of 367 kDa. NR from K. antarctica used both NADPH and NADH, whereas NR from C. sorokiniana used NADH only. Both NRs used reduced methyl viologen (MVH) or benzyl viologen (BVH). In crude extracts, maximal NADH and MVH-dependent activities of cryophilic NR were found at 15 and 35 ∞ C, respectively, and retained 77 and 62% of maximal activity, respectively, at 10 ∞ C. Maximal NADH and MVH-dependent activities of mesophilic NR, however, were found at 25 and 45 ∞ C, respectively, with only 33 and 23% of maximal activities being retained at 10 ∞ C. In presence of 2 m M flavin adenine dinucleotide (FAD), activities of cryophilic NADH:NR and mesophilic NADH:NR were stable up to 25 and 35 ∞ C, respectively. Arrhenius plots constructed with cryophilic and mesophilic MVH:NR rate constants, in both presence or absence of FAD, showed break points at 15 and 25 ∞ C, respectively. Essentially, similar results were obtained for purified enzymes and for activities measured in crude extracts. Factors by which the rate increases by raising temperature 10 ∞ C ( Q 10 ) and apparent activation energy ( E a ) values for NADH and MVH activities measured in enzyme preparations without added FAD differed slightly from those measured with FAD. Overall thermal features of the NADH and MVH activities of the cryophilic NR, including optimal temperatures, heat inactivation (with/without added FAD) and break-point temperature in Arrhenius plots, are all shifted by about 10 ∞ C towards lower temperatures than those of the mesophilic enzyme. Transfer of electrons from NADH to nitrate occurs via all three redox centres within NR molecule, whereas transfer from MVH requires Mo-pterin prosthetic group only; therefore, our results strongly suggest that structural modification(s) for cold adaptation affect thermodynamic properties of each of the functional domains within NR holoenzyme in equal measure.

Temperature responses of growth, photosynthesis, fatty acid and nitrate reductase in Antarctic and temperate Stichococcus

“…NJ-10 and NJ-17 exhibited the maximum activity at 20°C in vitro, with 15 and 26 nmol NO 2 -min -1 mg -1 protein, respectively, while the maximum activity of nitrate reductase was found at 25°C with a value of 13 nmol NO 2 -min -1 mg -1 protein in FACHB753. Our results indicate that the two Antarctic algae, especially NJ-17, which shows a high level of nitrate reductase specific activity, share same properties which are typical of coldadapted organisms (Feller and Gerday 1997;Vona et al 2004;Di Martino Rigano et al 2006).…”

“…Many concerns have been concentrated on the polar psychrophiles and few reports on the psychrotolerants can be found. In comparison with mesophiles, the content of the poly-unsaturated and short chain fatty acids in the membrane lipid is much higher, the optimal enzyme activity of nitrate reductase, glutathione reductase, argininosuccinate lyase shifts toward lower temperatures and rates of photosynthesis and respiration show a higher value in polar psychrophiles (Vona et al 2004;Di Martino Rigano et al 2006;Morgan-Kiss et al 2006;Ding et al 2007). In contrast, Morgan-Kiss et al (2008) found that the composition of the membrane lipid in psychrophilic Chlorella BI was similar to that in mesophilic one and no short chain fatty acids were detected.…”

Alternative Cold Response Modes in Chlorella (Chlorophyta, Trebouxiophyceae) from Antarctica

“…Microalgae from this region can grow at low temperatures close to the freezing point (Morgan-Kiss et al 2006), depending on the synthesis of polyunsaturated fatty acids (Mock & Kroon 2002a, b;Morgan-Kiss et al 2002), cold-adapted nitrate reductase (Vona et al 2004;Di Martino Rigano et al 2006), and possibly the production of NO (Estevez & Puntarulo 2005). Compared to studies of their growth at low temperature, the antifreeze mechanisms of Antarctic microalgae are little known.…”

Cytomorphological and nitrogen metabolic enzyme analysis of psychrophilic and mesophilic Nostoc sp.: a comparative outlook

“…5d). The characteristics of NR from various algae belonging to different thermal habitat showed a wide range of temperature adaptation (Di Martino Rigano et al 2006). It is reported that the active centre of the enzyme gets structurally altered to shift the enzyme activity optimum to the region of higher temperatures and increased the thermal stability of the NR (Morozkina and Zvyagilskaya 2007).…”

“…Decreased enzyme activity at lower temperature may be due to the fact that the affinity for the substrate is greatly decreased at lower temperature (Reay et al 1999). Di Martino Rigano et al (2006) reported that NR enzyme from the psychrophilic algae Koliella antarctica possesses structural modifications that make it colder adapted, being more catalytically efficient at lower temperatures, but they are less thermally stable than NR from mesophilic Chlorella sorokiana simultaneously.…”