Catalytic properties of domain-exchanged chimeric proteins between firefly luciferase and Drosophila fatty Acyl-CoA synthetase CG6178

Firefly luciferase and fatty acyl-CoA synthetase are members of the acyl-CoA synthetase super family, which consists of a large N-terminal domain and a small C-terminal domain. Previously we found that firefly luciferase has fatty acyl-CoA synthetic activity, and also identified that the homolog of firefly luciferase in Drosophila melanogaster (CG6178) is a fatty acyl-CoA synthetase and is not a luciferase. In this study, we constructed chimeric proteins by exchanging the domain between Photinus pyralis luciferase (PpLase) and Drosophila CG6178, and determined luminescence and fatty acyl-CoA synthetic activities. A chimeric protein with the N-terminal domain of PpLase and the C-terminal domain of CG6178 (Pp/Dm) had luminescence activity, showing approximately 4% of the activity of wild-type luciferase. The Pp/Dm protein also had fatty acyl-CoA synthetic activity and the substrate specificity was similar to PpLase. In contrast, a chimeric protein with the N-terminal domain of CG6178 and the C-terminal of PpLase (Dm/Pp) had only fatty acyl-CoA synthetase activity, and the substrate specificity was similar to CG6178. These results suggest that the N-terminal domain of firefly luciferase is essential for substrate recognition, and that the C-terminal domain is indispensable but not specialized for the luminescence reaction.     

Hatena arenicola gen. et sp. nov., a katablepharid undergoing probable plastid acquisition

Hatena arenicola gen. et sp. nov., an enigmatic flagellate of the katablepharids, is described. It shows ultrastructural affinities to the katablepharids, including large and small ejectisomes, cell covering, and a feeding apparatus. Although molecular phylogenies of the 18S ribosomal DNA support its classification into the katablepharids, the cell is characterized by a dorsiventrally compressed cell shape and a crawling motion, both of which are unusual within this group. The most distinctive feature of Hatena arenicola is that it harbors a Nephroselmis symbiont. This symbiosis is distinct from previously reported cases of ongoing symbiosis in that the symbiont plastid is selectively enlarged, while other structures such as the mitochondria, Golgi body, cytoskeleton, and endomembrane system are degraded; the host and symbiont have developed a morphological association, i.e., the eyespot of the symbiont is always at the cell apex of Hatena arenicola; and only one daughter cell inherits the symbiont during cell division, resulting in a symbiont-bearing green cell and a symbiont-lacking colorless cell. Interestingly, the colorless cells have a feeding apparatus that corresponds to the location of the eyespot in symbiont-bearing cells, and they are able to feed on prey cells. This indicates that the morphology of the host depends on the presence or absence of the symbiont. These observations suggest that Hatena arenicola has a unique "half-plant, half-predator" life cycle; one cell divides into an autotrophic cell possessing a symbiotic Nephroselmis species, and a symbiont-lacking colorless cell, which later develops a feeding apparatus de novo. The evolutionary implications of Hatena arenicola as an intermediate step in plastid acquisition are discussed in the context of other examples of ongoing endosymbioses in dinoflagellates.     

Effects of water-level fluctuations on the arbuscular mycorrhizal colonization of Typha latifolia L.

Flooding has been described as one of the primary factors affecting arbuscular mycorrhizal (AM) colonization in wetlands. We investigated the effect of water-level fluctuations on AM colonization of Typha latifolia L. using an experimental wetland in southeastern Idaho, USA that received intermittent flows. Unlike previous research that has examined the effect of flooding on AM fungi using topographic gradients, we replicated flooding in time by sampling across multiple flooding events. AM colonization of T. latifolia occurred during flooded and unflooded periods, but was markedly reduced at drawdown. Both hyphal (R = 0.74, P = 0.015) and arbuscular (R = 0.67, P = 0.033) colonization were positively correlated with the length of the unflooded period. Taken together, the length of the unflooded period and soil moisture explained 83% of the variation in mean hyphal colonization (R² = 0.83, P = 0.001). Overall, the results of this investigation show that drawdown represents a period of reduced AM colonization in T. latifolia.     

Engineering of the pH-dependence of thermolysin activity as examined by site-directed mutagenesis of Asn112 located at the active site of thermolysin

Asn112 is located at the active site of thermolysin, 5-8 A from the catalytic Zn2+ and catalytic residues Glu143 and His231. When Asn112 was replaced with Ala, Asp, Glu, Lys, His, and Arg by site-directed mutagenesis, the mutant enzymes N112D and N112E, in which Asn112 is replaced with Asp and Glu, respectively, were secreted as an active form into Escherichia coli culture medium, while the other four were not. In the hydrolysis of a neutral substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide, the kcat/Km values of N112D and N112E exhibited bell-shaped pH-dependence, as did the wild-type thermolysin (WT). The acidic pKa of N112D was 5.7 +/- 0.1, higher by 0.4 +/- 0.2 units than that of WT, suggesting that the introduced negative charge suppressed the protonation of Glu143 or Zn2+-OH. In the hydrolysis of a negatively charged substrate, N-carbobenzoxy-l-Asp-l-Phe methyl ester (ZDFM), the pH-dependence of kcat/Km of the mutants decreased with increase in pH from 5.5 to 8.5, while that of WT was bell-shaped. This difference might be explained by the electrostatic repulsion between the introduced Asp/Glu and ZDFM, suggesting that introducing ionizing residues into the active site of thermolysin might be an effective means of modifying its pH-activity profile.     

Blue fluorescent protein from the calcium-sensitive photoprotein aequorin: catalytic properties for the oxidation of coelenterazine as an oxygenase

Blue fluorescent protein from the calcium-binding photoprotein aequorin (BFP-aq) is a complex of Ca2+ -bound apoaequorin and coelenteramide, and shows luminescence activity like a luciferase, catalyzing the oxidation of coelenterazine with molecular oxygen. To understand the catalytic properties of BFP-aq, various fluorescent proteins (FP-aq) have been prepared from semi-synthetic aequorin and characterized in comparison with BFP-aq. FP-aq has luciferase activity and could be regenerated into native aequorin by incubation with coelenterazine. The results from substrate specificity studies of FP-aq using various coelenterazine analogues have suggested that the oxidation of coelenterazine by BFP-aq in the luciferase reaction and the regeneration process to aequorin might involve the same catalytic site of BFP-aq.