Assessing the Impacts of Experimentally Elevated Temperature on the Biological Composition and Molecular Chaperone Gene Expression of a Reef Coral

Due to the potential for increasing ocean temperatures to detrimentally impact reef-building corals, there is an urgent need to better understand not only the coral thermal stress response, but also natural variation in their sub-cellular composition. To address this issue, while simultaneously developing a molecular platform for studying one of the most common Taiwanese reef corals, Seriatopora hystrix, 1,092 cDNA clones were sequenced and characterized. Subsequently, RNA, DNA and protein were extracted sequentially from colonies exposed to elevated (30°C) temperature for 48 hours. From the RNA phase, a heat shock protein-70 (hsp70)-like gene, deemed hsp/c, was identified in the coral host, and expression of this gene was measured with real-time quantitative PCR (qPCR) in both the host anthozoan and endosymbiotic dinoflagellates (genus Symbiodinium). While mRNA levels were not affected by temperature in either member, hsp/c expression was temporally variable in both and co-varied within biopsies. From the DNA phase, host and Symbiodinium hsp/c genome copy proportions (GCPs) were calculated to track changes in the biological composition of the holobiont during the experiment. While there was no temperature effect on either host or Symbiodinium GCP, both demonstrated significant temporal variation. Finally, total soluble protein was responsive to neither temperature nor exposure time, though the protein/DNA ratio varied significantly over time. Collectively, it appears that time, and not temperature, is a more important driver of the variation in these parameters, highlighting the need to consider natural variation in both gene expression and the molecular make-up of coral holobionts when conducting manipulative studies. This represents the first study to survey multiple macromolecules from both compartments of an endosymbiotic organism with methodologies that reflect their dual-compartmental nature, ideally generating a framework for assessing molecular-level changes within corals and other endosymbioses exposed to changes in their environment

Functional roles of arginine residues in mung bean vacuolar H+-pyrophosphatase

AbstractPlant vacuolar H+-translocating inorganic pyrophosphatase (V-PPase EC 3.6.1.1) utilizes inorganic pyrophosphate (PPi) as an energy source to generate a H+ gradient potential for the secondary transport of ions and metabolites across the vacuole membrane. In this study, functional roles of arginine residues in mung bean V-PPase were determined by site-directed mutagenesis. Alignment of amino-acid sequence of K+-dependent V-PPases from several organisms showed that 11 of all 15 arginine residues were highly conserved. Arginine residues were individually substituted by alanine residues to produce R→A-substituted V-PPases, which were then heterologously expressed in yeast. The characteristics of mutant variants were subsequently scrutinized. As a result, most R→A-substituted V-PPases exhibited similar enzymatic activities to the wild-type with exception that R242A, R523A, and R609A mutants markedly lost their abilities of PPi hydrolysis and associated H+-translocation. Moreover, mutation on these three arginines altered the optimal pH and significantly reduced K+-stimulation for enzymatic activities, implying a conformational change or a modification in enzymatic reaction upon substitution. In particular, R242A performed striking resistance to specific arginine-modifiers, 2,3-butanedione and phenylglyoxal, revealing that Arg242 is most likely the primary target residue for these two reagents. The mutation at Arg242 also removed F− inhibition that is presumably derived from the interfering in the formation of substrate complex Mg2+–PPi. Our results suggest accordingly that active pocket of V-PPase probably contains the essential Arg242 which is embedded in a more hydrophobic environment

The function of Lysine 425 of phosphoglucose isomerase from Bacillus Stearothermophilus

中 文 摘 要 磷酸葡萄糖異構( phosphoglucose isomerase EC 5.3.1.9﹔簡稱PGI)為醛糖-酮醣異構 之一種，其主要功能是催化磷酸六碳糖(phosphate hexose)的異構化作 用(isomerization)，為糖解作用及生糖作用之重要中間代謝酵素。依據 目前磷酸葡萄糖異構的催化機制模型中，磷酸六碳糖會先行開環作用再 行異構化作用。此種開環的作用可能由一個質子化之離胺酸(lysine)上 ε-銨基(ε-ammonium)側鏈參與磷酸六碳糖之開環作用。利用定點突變技 術來研究蛋白質的結構與功能是非常有用的研究工具，本研究是利用此種 技術來探討B. stearothermophilus兩種同功(isoenzyme) PGIA及PGIB 之保留性胺基酸殘基的功能。在PGIA中高度保留性胺基酸序列之離胺酸 Lys 425位置，以聚合鏈鎖反應(PCR)定點突變法行之﹐在PGIB上保留性 胺基酸殘基組胺酸His 307位置，以單股DNA之定點突變法行之。經定點突 變後產生的結果中，我們選擇PGI A Lys 425→Ala, Asp, Phe, Trp, Val 等5株定點突變株進行大量表現及純化。經由酵素動力學分析並與野生株 進行比較，發現在KM值上Lys 425定點突變株影響很小，在0.4到1.3倍之 間。在k cat值上Lys 425定點突變株影響很大有102倍的下降。由以上結 果中，可見Lys 425之定點突變主要的影響在k cat值上，但在KM值上並沒 有很明顯的影響，且在k cat/ KM值上亦有明顯下降的改變。 由此結果中 我們認為Lys 425在磷酸葡萄糖異構之酵素催化機制中，可能參與在酵 素-受質複合物之過渡態(transition state)的穩定。另由活化自由能差( ΔΔG?的計算中，其能量改變約3.5~2.4 kcal/mole，據此改變量推估 PGIA Lys 425在酵素-受質複合物之過渡態(transition state)的穩定上 ﹐可能提供一個離子化氫鍵的穩定力量。在Lys 425→Asp之突變分析結果 發現胺基酸殘基側鏈的電性改變，並沒有引起更大的活化能之自由能差( ΔΔG?的改變，此結果顯然Lys 425並非以電荷吸引力(electrostatic interaction)的形式參與酵素-受質複合物的過渡態穩定。AbstractPhosphoglucose isomerase (EC 5.1.3.9) is an aldose- ketose isomerase that catalyzes the isomerization between glucose-6-phosphate and fructose-6-phosphate and functions in the pathway of glycolysis and gluconeogenesis. Although the genes coding for phosphoglucose isomerase have been cloned from at least twenty organisms, the catalytic mechanism in terms of structure-function relationship is far from understood. From the alignment of the deduced amino acid sequences of phosphoglucose isomerases, two conserved regions have been identified; they are[DENS]-x-[LIVM]-G-G-R-[FY]-S-[LIVM]-x-[STA]-[PAS]-[LIVM]-G, and G-x-[LIVM]- [LIVMFYW]-x(4)-[FY]-[DN]-Q-x-G-V-E-x(2)-K. This study intends to investigate the importance of the lysine within the second conserved region. To do that the corresponding lysine (Lys 425) from one of the phosphoglucose isomerases (PGIA) of Bacillus stearothermophilus was changed to alanine, valine, phenylalanine, aspartate, and tryptophan. The kinetic data show there is no significant change in the Michaelis constant (KM) upon the mutation of Lys 425; whereas, the catalytic constant (kcat) decreases several-hundred folds. Such changes indicate that the major function of Lys 425 is to stabilize the transition-state rather than the ground-state of substrate. The side-chain of Lys 425 was estimated to contribute a binding energy of 3.5-2.4 kcal/mole to stabilize the transition-state based on the calculationΔΔG?= -RT ln {(kcat/KM)mut/(kcat/KM) wild-type}