Lysine 206 in <i>Arabidopsis</i> phytochrome A is the major site for ubiquitin-dependent protein degradation

Rattanapisit, Kaewta,Cho, Man-Ho,Bhoo, Seong Hee Japanese Biochemical Society 2016 The Journal of biochemistry Vol.159 No.2

<P>Phytochrome A (phyA) is a light labile phytochrome that mediates plant development under red/far-red light condition. Degradation of phyA is initiated by red light-induced phyA-ubiquitin conjugation through the 26S proteasome pathway. The N-terminal of phyA is known to be important in phyA degradation. To determine the specific lysine residues in the N-terminal domain of phyA involved in light-induced ubiquitination and protein degradation, we aligned the amino acid sequence of the N-terminal domain of Arabidopsis phyA with those of phyA from other plant species. Based on the alignment results, phytochrome over-expressing Arabidopsis plants were generated. In particular, wild-type and mutant (substitutions of conserved lysines by arginines) phytochromes fused with GFP were expressed in phyA(-)211 Arabidopsis plants. Degradation kinetics of over-expressed phyA proteins revealed that degradation of the K206R phyA mutant protein was delayed. Delayed phyA degradation of the K206R phyA mutant protein resulted in reduction of red-light-induced phyA-ubiquitin conjugation. Furthermore, seedlings expressing the K206R phyA mutant protein showed an enhanced phyA response under far-red light, resulting in inhibition of hypocotyl elongation as well as cotyledon opening. Together, these results suggest that lysine 206 is the main lysine for rapid ubiquitination and protein degradation of Arabidopsis phytochrome A.</P>

(Regular Papers) Purification and Characterization of a Copper-Containing Amine Oxidase from Mycobacterium Sp. Strain JC1 DSM 3803 Grown on Benzylamine

Lee, Hyun-Il,Kim, Young Min,Ro, Young Tae Japanese Biochemical Society 2008 The Journal of biochemistry Vol.144 No.1

A bacterial semicarbazide-sensitive amine oxidase (SSAO) was purified and characterized from Mycobacterium sp. strain JC1 DSM 3803 grown on benzylamine. During the purification procedures, the enzyme was tending to aggregate and exhibited heterogeneity in native PAGE. The heterogeneous forms having amine oxidase (AO) activity could be separated by their native molecular weights using gel-filtration chromatography. Most of the AOs behaved as dimers (M_r 150,000) composed of a 75-kDa subunit, but some aggregated to form tetramers (M_r 300,000). Besides their native molecular weight, subunit composition and V_max value, both forms (dimer and tetramer) have almost identical biochemical properties (e.g. subunit size, optimum pH and temperature, activation energy, K_m value on benzylamine, substrate and inhibitor specificities). When AO activity was observed by activity staining, the best-oxidized substrate was benzylamine, although the AO also oxidized tyramine and histamine. The AO was strongly inhibited by semicarbazide and isoniazid, but KCN did not affect its activity. The purified enzyme was shown to contain 2.39 mol of copper per mole of subunit, but there were no evidences of topaquinone co-factor involvement, when tested by absorption spectrum analysis and redox-cycling staining for quinoprotein detection.

Cloning and Characterization of Phosphoglucomutase and Phosphomannomutase Derived from Sphingomonas chungbukensis DJ77

Yoon, Sung-Sook,Park, Sung-Ha,Kim, Young-Chang,Shin, Malshik,Chong, Chom-Kyu,Choi, Jung-Do Japanese Biochemical Society 2008 The Journal of biochemistry Vol.144 No.4

The enzymes phosphoglucomutase (PGM) and phosphomannomutase (PMM) play an important role in the synthesis of extracellular polysaccharide. By colony hybridization of the fosmid library of Sphingomonas chungbukensis DJ77, an open reading frame (ORF-1) of 1,626 nucleotides, whose predicted product is highly homologous with other PGM proteins from several bacterial species, was identified. An additional open reading frame (ORF-2) of 1,437 nucleotides was identified, and its encoded protein shows a high level of similarity with the PGM/PMM protein family. The two genes were cloned into a bacterial expression vector pET-15b (+) and expressed in Escherichia coli as fusion proteins with (His)6-tag. Both recombinant proteins (designated as SP-1 and SP-2 for ORF-1 and ORF-2, respectively) exhibited PGM and PMM activities. The molecular masses of subunits of SP-1 and SP-2 were estimated to be around 58 and 51 kDa from SDS?PAGE, respectively. However, molecular masses of SP-1 and SP-2 in their native condition were determined to be approximately 59.5 and 105.4 kDa, according to non-denaturing PAGE, respectively. The SP-1 protein has a preference for glucose-1-phosphate rather than mannose-1-phosphate, while the preferred substrate of SP-2 is mannose-1-phosphate. Thus, the existence of two proteins with bifunctional PGM/PMM activities was first found S. chungbukensis DJ77.

Structural mechanism of disulphide bond-mediated redox switches.

Japanese Biochemical Society 2012 The Journal of biochemistry Vol.151 No.6

<P>The oxidation of cysteine sulphydryl in proteins produces sulphenic acid that can form a reversible disulphide bond with another cysteine. The disulphide bond formation often triggers switches in protein structure and activity, especially when the distance between the two cysteine sulphur atoms is longer than the resulting disulphide bond distance. As an early example for the reversible disulphide bond-mediated functional switches, the reduced and oxidized forms of the bacterial transcription factor OxyR were characterized by X-ray crystallography. Recently, the Drosophila vision signalling protein, the association of inactivation-no-afterpotential D (INAD) was analysed by structural and functional methods. The two conserved cysteines of INAD were found to cycle between reduced and oxidized states during the light signal processing in Drosophila eyes, which was achieved by conformation dependent modulation of the disulphide bond redox potential. The production of the hypertension control peptide angiotensins was also shown to be controlled by the reversible disulphide bond in the precursor protein angiotensinogen. The crystal structure of the complex of angiotensiongen with its processing enzyme renin elucidated the role of the disulphide bond in stabilizing the precursor-enzyme complex facilitating the production of angiotensins. The increasing importance of the disulphide bond-mediated redox switches in normal and diseased states has implications in the development of novel antioxidant-based therapeutic approaches.</P>

Apolipoprotein A-IV is a novel substrate for matrix metalloproteinases.

Park, Ji Yoon,Park, Jun Hyoung,Jang, Wookju,Hwang, In-Kwan,Kim, In Ja,Kim, Hwa-Jung,Cho, Kyung-Hyun,Lee, Seung-Taek Japanese Biochemical Society 2012 The Journal of biochemistry Vol.151 No.3

<P>Screening of matrix metalloproteinase (MMP)-14 substrates in human plasma using a proteomics approach previously identified apolipoprotein A-IV (apoA-IV) as a novel substrate for MMP-14. Here, we show that among the tested MMPs, purified apoA-IV is most susceptible to cleavage by MMP-7, and that apoA-IV in plasma can be cleaved more efficiently by MMP-7 than MMP-14. Purified recombinant apoA-IV (44-kDa) was cleaved by MMP-7 into several fragments of 41, 32, 29, 27, 24, 22 and 19 kDa. N-terminal sequencing of the fragments identified two internal cleavage sites for MMP-7 in the apoA-IV sequence, between Glu(185) and Leu(186), and between Glu(262) and Leu(263). The cleavage of lipid-bound apoA-IV by MMP-7 was less efficient than that of lipid-free apoA-IV. Further, MMP-7-mediated cleavage of apoA-IV resulted in a rapid loss of its intrinsic anti-oxidant activity. Based on the fact that apoA-IV plays important roles in lipid metabolism and possesses anti-oxidant activity, we suggest that cleavage of lipid-free apoA-IV by MMP-7 has pathological implications in the development of hyperlipidemia and atherosclerosis.</P>

Low pH-driven folding of WW45-SARAH domain leads to stabilization of the WW45-Mst2 complex

Song, Jinsue,Hong, Hye Rim,Yamashita, Eiki,Park, Il Yeong,Lee, Soo Jae Japanese Biochemical Society 2015 The Journal of biochemistry Vol.158 No.3

<P>The scaffolding protein Salvador (Sav) plays a key role in the Hippo (Hpo) signalling pathway, which controls tissue growth by inhibiting cell proliferation and promoting apoptosis. Dysregulation of the Hippo pathway contributes to cancer development. Since the identification of the first <I>Sav</I> gene in 2002, very little is known regarding the molecular basis of Sav-SARAH mediating interactions due to its insolubility. In this study, refolding of the first Sav (known as WW45)-SARAH provided insight into the biochemical and biophysical properties, indicating that WW45-SARAH exhibits properties of a disordered protein, when the domain was refolded at a neutral pH. Interestingly, WW45-SARAH shows folded and rigid conformations relative to the decrease in pH. Further, diffracting crystals were obtained from protein refolded under acidic pH, suggesting that the refolded WW45 protein at low pH has a homogeneous and stable conformation. A comparative analysis of molecular properties found that the acidic-stable fold of WW45-SARAH enhances a heterotypic interaction with Mst2-SARAH. In addition, using an Mst2 mutation that disrupts homotypic dimerization, we showed that the monomeric Mst2-SARAH domain could form a stable complex of 1:1 stoichiometric ratio with WW45 refolded under acidic pH.</P>

Molecular characterization of acidic peptide:<i>N</i>-glycanase from the dimorphic yeast <i>Yarrowia lipolytica</i>

Lee, Kyung Jin,Gil, Jin Young,Kim, Sang-Yoon,Kwon, Ohsuk,Ko, Kisung,Kim, Dong-Il,Kim, Dae Kyong,Kim, Ha Hyung,Oh, Doo-Byoung Japanese Biochemical Society 2015 The Journal of biochemistry Vol.157 No.1

<P>Peptide:<I>N</I>-glycanase (PNGase) A is used preferentially to cleave the glycans from plant and insect glycopeptides. Although many putative PNGase A homologous genes have been found in the plant and fungus kingdoms through sequence similarity analyses, only several PNGases from plants and one from a filamentous fungus have been characterized. In this study, we identified and characterized a PNGase A-like enzyme, PNGase Yl, in the dimorphic yeast <I>Yarrowia lipolytica</I>. The corresponding gene was cloned and recombinantly expressed in <I>Pichia pastoris</I>. The purified enzyme cleaved glycans from glycopeptides with the maximum activity at pH 5. No metal ions were required for full activity, and rather it was repressed by three metal ions (Fe<SUP>3+</SUP>, Cu<SUP>2+</SUP> and Zn<SUP>2+</SUP>). Using glycopeptide substrates, PNGase Yl was shown to release various types of <I>N</I>-glycans including high-mannose and complex-type glycans as well as glycans containing core-linked α(1,3)-fucose that are frequently found in plants and insects. Moreover, in comparison with PNGase A, PNGase Yl was able to cleave with higher efficiency the glycans from some denatured glycoproteins. Taken together, our results suggest that PNGase Yl, the first biochemically characterized yeast PNGase A homologue, can be developed through protein engineering as a useful deglycosylation tool for <I>N</I>-glycosylation study.</P>

Nuclear import of human histone lysine-specific demethylase LSD1

Jin, Yanhua,Kim, Tae Young,Kim, Min Seong,Kim, Min Aeh,Park, Su Hyung,Jang, Yeun Kyu Japanese Biochemical Society 2014 The Journal of biochemistry Vol.156 No.6

<P>Upregulation and nuclear retention of the human histone demethylase LSD1 are correlated with aggressiveness and poor outcome of several cancer types, but the molecular mechanism of LSD1 nuclear import remains unclear. Here, we found that the N-terminal flexible region of LSD1 contains a nuclear localization signal (NLS), <SUP>112</SUP>RRKRAK<SUP>117</SUP>. Mutation or deletion of the NLS completely abolished the nuclear import of LSD1, suggesting the motif is a <I>bona fide</I> NLS. More importantly, our GST pull-down assay showed that LSD1 physically interacts with three proteins of importin α family. In addition, our data suggest that the nuclear localization of LSD1 via the NLS is not a cell-type specific event. Thus, these findings demonstrate for the first time that the NLS motif within the N-terminal flexible domain of LSD1 is critical for its nuclear localization via interaction with importin α proteins.</P>

Prodomain Processing of Recombinant Plasmepsin II and IV, the Aspartic Proteases of Plasmodium falciparum, Is Auto- and Trans-Catalytic

Man Kim, Yong,Hyang Lee, Mi,Gwang Piao, Tae,Wha Lee, John,Ho Kim, Jong,Lee, SungGa,Min Choi, Kyung,Hua Jiang, Jing,Ue Kim, Tae,Park, Hyun Japanese Biochemical Society 2006 The Journal of biochemistry Vol.139 No.2

<P>Prodomain processing of the four food vacuole plasmepsins (PMs), the malarial aspartic proteases, is prerequisite for their activity on hemoglobin degradation of the parasite Plasmodium falciparum. Although previous studies have suggested the involvement of a calpain-like PM convertase in the processing of PMs, the underlying mechanism of their processing remains to be clarified. Here, to investigate the mechanism by which food vacuole PM II and IV are processed, we used their wild-type and mutant proteins in which the catalytic Asp residue in two active-site motifs was mutated, as well as protease inhibitors. Autocatalytic processing of wild-type PM II and IV was inhibited only by an aspartic protease inhibitor pepstatin A. Unexpectedly, their proteolytic activities were inhibited not only by pepstatin A but also by calpain inhibitor ALLN. The active-site mutants of both PM II and IV showed neither autocatalytic processing nor proteolytic activities. However, the mutants of both PMs were efficiently processed upon incubation with their respective wild type proteins. Furthermore, the mutants of both PMs were processed upon incubation with each other's wild-type PM in both pepstatin A- and ALLN-sensitive manners. These results suggest that the processing of PM II and IV occurs via an intra- and inter-molecular autocatalytic event as well as via a transcatalytic event between them.</P>

Catalytic Promiscuity in Dihydroxy-Acid Dehydratase from the Thermoacidophilic Archaeon Sulfolobus solfataricus

Kim, Seonghun,Bok Lee, Sun Japanese Biochemical Society 2006 The Journal of biochemistry Vol.139 No.3

<P>Dihydroxy-acid dehydratase (DHAD) is one of the key enzymes involved in the biosynthetic pathway of the branched chain amino acids. Although the enzyme has been purified and characterized in various mesophiles, including bacteria and eukarya, the biochemical properties of DHAD from hyperthermophilic archaea have not yet been reported. In this study we cloned, expressed in Escherichia coli, and purified a DHAD homologue from the thermoacidophilic archaeon Sulfolobus solfataricus, which grows optimally at 80°C and pH 3. The recombinant S. solfataricus DHAD (rSso_DHAD) showed the highest activity on 2,3-dihydroxyisovalerate among 17 aldonic acids tested. Interestingly, this enzyme also displayed high activity toward D-gluconate and some other pentonic and hexonic sugar acids. The kcat/Km values were 140.3 mM?1 s?1 for 2,3-dihydroxyisovalerate and 20.0 mM?1 s?1 for D-gluconate, respectively. A possible evolutionary explanation for substrate promiscuity was provided through amino acid sequence alignments of DHADs and 6-phosphogluconate dehydratases from archaea, bacteria and eukarya.</P>