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Succinic semialdehyde dehydrogenase (SSADH) catalyses the conversion of succinic semialdehyde into succinic acid and two electrons are transferred to NAD(P)+ to yield NAD(P)H. Our previous work has already reported the catalytic role of Cys289 of two‐cysteine SSADH from Acinetobacter baumannii (AbSSADH). However, the mechanistic role of the neighbouring conserved Cys291 and Glu255 remains unexplored. In this study, the functional roles of Cys291 and Glu255 in AbSSADH catalysis have been characterized. Results demonstrated that the E255A activity was almost completely lost, ~ 7000‐fold lower than the wild‐type (WT), indicating that Glu255 is very crucial and directly involved in AbSSADH catalysis. However, the C291A and C291S variants activity and catalytic turnover (kcat) decreased ~ 2‐fold and 9‐fold respectively. To further characterize the functional roles of Cys291, we employed two pH‐dependent methods; pre‐steady‐state burst amplitude and NADP–enzyme adduct formation. The results showed that the pKa values of catalytic Cys289 measured for the WT and C291A reactions were 7.8 and 8.7–8.8, respectively, suggesting that Cys291 can lower the pKa of Cys289 and consequently trigger the deprotonation of a Cys289 thiol. In addition, the Cys291 also plays a role in disulfide/sulfhydryl redox regulation for AbSSADH activity. Hence, we demonstrated for the first time the dual functions of Cys291 in enhancing the nucleophilicity of the catalytic Cys289 and regulating a disulfide/sulfhydryl redox switch for AbSSADH catalysis. The mechanistic insights into the nucleophilicity enhancement of the catalytic cysteine of AbSSADH might be applicable to understanding how the microenvironment increases cysteine reactivity in other enzymes in the aldehyde dehydrogenase superfamily. [ABSTRACT FROM AUTHOR]

Neuropeptide F (NPF) plays critical roles in controlling the feeding and reproduction of prawns. In the present study, we investigated changes in the expression levels of Macrobrachium rosenbergii neuropeptide F (MrNPF), and its neuroanatomical distribution in eyestalk (ES), brain (BR), subesophageal ganglion (SEG), thoracic ganglia (TG), and abdominal ganglia (AG), during the ovarian cycle of female prawn. By qRT‐PCR, the amount of MrNPF transcripts exhibited a gradual increase in the ES, BR, and combined SEG and TG from stages I and II, to reach a maximum level at stage III, and slightly declined at stage IV, respectively. The highest to lowest expression levels were detected in combined SEG and TG, BR, ES, and AG, respectively. MrNPF immunolabeling was observed in several neuronal clusters, associated fibers, and neuropils of these central nervous system (CNS) tissues. MrNPF‐ir was more intense in neurons and neuropils of SEG and TG than those found in other parts of the CNS. The number of MrNPF‐ir neurons and intensity of MrNPF‐ir were higher in the ES, BR, SEG, and TG at the late stages than those at the early stages of the ovarian cycle, while those in AG exhibited insignificant change. Taken together, there is a correlation between changes in the neuroanatomical distribution of MrNPF and stages of the ovarian cycle, implying that MrNPF may be an important neuropeptide that integrates sensory stimuli, including photo‐, chemo‐, and gustatory receptions, to control feeding and reproduction, particularly ovarian development, of this female prawn, M. rosenbergii. [ABSTRACT FROM AUTHOR]

Abstract Background The distinct differences in gene control mechanisms acting in the nucleus between Plasmodium falciparum and the human host could lead to new potential drug targets for anti-malarial development. New molecular toolkits are required for dissecting molecular machineries in the P. falciparum nucleus. One valuable tool commonly used in model organisms is protein targeting to specific sub-cellular locations. Targeting proteins to specified locations allows labeling of organelles for microscopy, or testing of how the protein of interest modulates organelle function. In recent years, this approach has been developed for various malaria organelles, such as the mitochondrion and the apicoplast. A tool for targeting a protein of choice to the P. falciparum nucleus using an exogenous nuclear localization sequence is reported here. Methods To develop a nuclear targeting system, a putative nuclear localization sequence was fused with green fluorescent protein (GFP). The nuclear localization sequence from the yeast transcription factor Gal4 was chosen because of its well-defined nuclear localization signal. A series of truncated Gal4 constructs was also created to narrow down the nuclear localization sequence necessary for P. falciparum nuclear import. Transfected parasites were analysed by fluorescent and laser-scanning confocal microscopy. Results The nuclear localization sequence of Gal4 is functional in P. falciparum. It effectively transported GFP into the nucleus, and the first 74 amino acid residues were sufficient for nuclear localization. Conclusions The Gal4 fusion technique enables specific transport of a protein of choice into the P. falciparum nucleus, and thus provides a tool for labeling nuclei without using DNA-staining dyes. The finding also indicates similarities between the nuclear transport mechanisms of yeast and P. falciparum. Since the nuclear transport system has been thoroughly studied in yeast, this could give clues to research on the same mechanism in P. falciparum.

Bacterial luciferase catalyzes a bioluminescent reaction by oxidizing long‐chain aldehydes to acids using reduced FMN and oxygen as co‐substrates. Although a flavin C4a‐peroxide anion is postulated to be the intermediate reacting with aldehyde prior to light liberation, no clear identification of the protonation status of this intermediate has been reported. Here, transient kinetics, pH variation, and site‐directed mutagenesis were employed to probe the protonation state of the flavin C4a‐hydroperoxide in bacterial luciferase. The first observed intermediate, with a λmax of 385 nm, transformed to an intermediate with a λmax of 375 nm. Spectra of the first observed intermediate were pH‐dependent, with a λmax of 385 nm at pH < 8.5 and 375 at pH > 9, correlating with a pKa of 7.7–8.1. These data are consistent with the first observed flavin C4a intermediate at pH < 8.5 being the protonated flavin C4a‐hydroperoxide, which loses a proton to become an active flavin C4a‐peroxide. Stopped‐flow studies of His44Ala, His44Asp, and His44Asn variants showed only a single intermediate with a λmax of 385 nm at all pH values, and none of these variants generate light. These data indicate that His44 variants only form a flavin C4a‐hydroperoxide, but not an active flavin C4a‐peroxide, indicating an essential role for His44 in deprotonating the flavin C4a‐hydroperoxide and initiating chemical catalysis. We also investigated the function of the adjacent His45; stopped‐flow data and molecular dynamics simulations identify the role of this residue in binding reduced FMN. [ABSTRACT FROM AUTHOR]