Noriyuki Fukunaga

Structure of the Monomeric Isocitrate Dehydrogenase: Evidence of a Protein Monomerization by a Domain Duplication

NADP(+)-dependent isocitrate dehydrogenase is a member of the beta-decarboxylating dehydrogenase family and catalyzes the oxidative decarboxylation reaction from 2R,3S-isocitrate to yield 2-oxoglutarate and CO(2) in the Krebs cycle. Although most prokaryotic NADP(+)-dependent isocitrate dehydrogenases (IDHs) are homodimeric enzymes, the monomeric IDH with a molecular weight of 80-100 kDa has been found in a few species of bacteria. The 1.95 A crystal structure of the monomeric IDH revealed that it consists of two distinct domains, and its folding topology is related to the dimeric IDH. The structure of the large domain repeats a motif observed in the dimeric IDH. Such a fusional structure by domain duplication enables a single polypeptide chain to form a structure at the catalytic site that is homologous to the dimeric IDH, the catalytic site of which is located at the interface of two identical subunits.

Membrane lipid composition and glucose uptake in two psychrotolerant bacteria from Antarctica

Summary: The thermal responses of membrane lipid composition and function in two bacterial species isolated recently from Heywood Lake sediment in Antarctica have been investigated. Both isolates are Gram-negative psychrotolerant (psychrotrophic) species growing well at 0 and 25 °C, but having very different optimum growth temperatures, of 9·7 °C for strain CR3/F/w/1/15 and 20·9 °C for strain CR3/F/w/2/10. The acyl lipids in strain 1/15 contain predominantly branched-chain fatty acids, mainly anteiso-15:0. The fatty acid composition and its response to temperature depends on the culture medium used. In nutrient-poor medium a wider range is found, including unsaturated fatty acids, which are absent when a nutrient-rich medium is used. A decrease in growth temperature produced a shortening of the average fatty acid chain length in rich medium, whereas in poor medium there was a decrease in straight-chain, saturated and unsaturated relative to branched-chain fatty acids. The acyl lipids of strain 2/10 contain 16:0 and 16:1 as the major fatty acids, particularly in rich medium, when they comprise > 90% of the total. Increasing proportions of 16:1 were converted to cyclopropane-17:0 at higher temperatures during growth in poor medium. A decrease in growth temperature in rich or poor medium resulted in an increase in the unsaturation index. When the bacteria were grown in rich or poor medium at 5 °C the strain with more ‘psychrophilic’ characteristics (1/15) took up glucose at a faster rate than did the strain with more ‘psychrotrophic’ characteristics (2/10), whereas when they were grown at 20 °C in either medium the rate of glucose uptake by strain 2/10 was generally faster than that of strain 1/15. Thus, when the bacteria were grown at a low temperature the glucose uptake system of the more ‘psychrophilic’ strain was better adapted to function at zero, compared with that of the more ‘psychrotrophic’ strain. The basis of this difference is not due to the lack of temperature-dependent fatty acid changes, as reported previously for some other cold-adapted bacteria.

Cloning, sequencing, and expression of a gene encoding the monomeric isocitrate dehydrogenase of the nitrogen-fixing bacterium, Azotobacter vinelandii.

Isocitrate dehydrogenase (IDH: EC 1.1.1.42) of Azotobacter vinelandii was purified to an electrophoretically homogeneous state, and a gene (icd) encoding this enzyme was cloned and sequenced. The N-terminal amino acid sequence of the purified enzyme was consistent with that deduced from the nucleotide sequence of the icd gene. The deduced amino acid sequence of this gene showed high identity (62–66%) to those of the other bacterial monomeric IDHs. Expression of the icd gene in Escherichia coli was examined by measuring the enzyme activity and mRNA level. Primer extension analyses revealed that two species of mRNAs with different lengths of 5′-untranslated regions (TS-1 and TS-2) were present, of which the 5′-terminals (TS-1 and TS-2 sites) were cytosines located at 244 bp and 101 bp upstream of translational initiation codon, respectively. Conserved promoter elements were present at —35 and —10 regions from the TS-1 site, whereas no such a common motif was found in the upstream region of the TS-2 site. Deletion of the promoter elements upstream of the TS-1 site resulted in complete loss of IDH activity in the E. coli transformant. When the promoter elements upstream of the TS-1 site were intact, the levels of TS-1 and TS-2 were varied greatly by altering exogenous nutrients for growth. The cells grown in a nutrient-rich medium produced large amounts of TS-1 and had a low level of IDH activity. In a nutrient-poor medium, the cells contained large amounts of TS-2 and high levels of IDH activity.

Purification and Characterization of a Cold-adapted Isocitrate Lyase and a Malate Synthase from Colwellia maris, a Psychrophilic Bacterium

Isocitrate lyase (ICL) and malate synthase (MS) of a psychrophilic marine bacterium, Colwellia maris, were purified to electrophoretically homogeneous state. The molecular mass of the ICL was found to be 240 kDa, composed of four identical subunits of 64.7 kDa. MS was a dimeric enzyme composed of 76.3 kDa subunits. N-Terminal amino acid sequences of the ICL and MS were analyzed. Purified ICL had its maximum activity at 20°C and was rapidly inactivated at the temperatures above 30°C, but the optimum temperature for the activity of MS was 45°C. NaCl was found to protect ICL from heat inactivation above 30°C, but the salt did not stabilize MS. Effects of temperatures on the kinetic parameters of both the enzymes were examined. The Km for the substrate (isocitrate) of ICL was decreased with decreasing temperature. On the other hand, the Km for the substrate (glyoxylate) of MS was increased with decreasing temperature. The calculated value of free energy of activation of ICL was on the same level as that of MS.

Crystallization and preliminary X-ray diffraction studies of monomeric isocitrate dehydrogenase by the MAD method using Mn atoms

NADP(+)-dependent isocitrate dehydrogenase (E.C. 1.1.1.42; IDH) is an enzyme of the Krebs cycle and catalyzes the oxidative decarboxylation reaction from DL-isocitrate to alpha-ketoglutarate, with a concomitant reduction of the coenzyme NADP(+) to NADPH. Single crystals of monomeric IDH from Azotobacter vinelandii in complex with DL-isocitrate and Mn(2+) were obtained by the hanging-drop vapour-diffusion method at room temperature. One crystal diffracted to a resolution of 2.9 A and was found to belong to the orthorhombic system; the space group was determined to be P2(1)2(1)2(1), with unit-cell parameters a = 108.4, b = 121.7, c = 129.7 A. The asymmetric unit contains two molecules of monomeric IDH, corresponding to a V(M) value of 2.66 A(3) Da(-1). The crystals were frozen in a capillary by a flash-cooling technique and MAD data were collected using Mn atoms as anomalous scatterers on beamline BL41XU at SPring-8, Japan. The positions of two Mn atoms binding to two independent IDH molecules were located from Bijvoet difference Patterson maps.

Bacterial Adaptation to Low Temperature:Implications for Cold-Inducible Genes

Escherichia coli and later found to be a cold-shock response common to many bacterial species. CspA of 7.4 kD, a major cold-shock protein in E. coli, has been shown to share structural similarity with a class of eukaryotic Y box proteins which have RNA-binding domains. Transient synthesis of CspA upon cold shock is mediated by increased stabilization of the mRNA at low temperatures. The proposed role of some cold-shock proteins including CspA in the bacterial adaptation to low temperatures is to function as a RNA chaperone in the regulation of translation. Some enzymes of psychrotrophic or psychrophilic bacteria exhibit unique features of a cold-adapted enzyme, high catalytic activity at a low temperature and rapid inactivation at a moderate temperature. A monomeric isocitrate dehydrogenase isozyme (IDH-II) of a psychrophilic bacterium, Vibrio sp. strain ABE-1, is a typical cold-adapted enzyme. In addition, this enzyme is induced at low temperatures. Low temperature-dependent expression of icdll encoding IDH-II is controlled by two different cis-elements located at the untranslated upstream region of the gene, one is a silencer and the other is essential for the low temperature response. The physiological role of IDH-II is evaluated by transforming E. coli with icdll. The growth rate of the E. coli transformants at low temperatures is dependent on the level of expressed IDH-II activity.

Purification and properties of NADP pyrophosphatase from Proteus vulgaris

Abstract 1. 1. NADP pyrophosphatase was purified about 700-fold from Proteus vulgaris by a procedure consisting of solubilization by detergents, fractionation by ammonium sulfate and cold ethanol, and column chromatography using DEAE-cellulose, hydroxylapatite and phosphocellulose. 2. 2. The purified enzyme was slightly activated by Mg 2+ and Mn 2+ and inhibited by Co 2+ and Zn 2+ . When the enzyme was treated with 5 mM EDTA and dialyzed against 10 mM Tris-HCl buffer, pH 7.0, the activity almost disappeared and the enzyme was reactivated strongly by Co 2+ and slightly by Mn 2+ . 3. 3. The optimum pH is 7.0. K m value for NADP + in Tris-HCl buffer at pH 7.0 was about 25 μM. 4. 4. Both NADP + and NADPH were cleaved rapidly at almost the same rate. NADH was also cleaved at half the rate of NADP + cleavage, while NAD + was cleaved at only 7% of this rate. ADP and UDP were also cleaved to release inorganic orthophosphate. 5. 5. NADP + cleavage by the enzyme was competitively inhibited by CoA, FAD, ATP, GTP, UTP and CTP. Among them CoA, GTP and CTP were very effective inhibitors. RNA, DNA and denatured DNA also inhibited NADP pyrophosphatase activity as well as boiled extracts of P. vulgaris , but these inhibitors were not competitive with NADP + .

NH4+ transport system of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1

Abstract NH4+ transport system of a psychrophilic marine bacterium Vibrio sp. strain ABE-1 (Vibrio ABE-1) was examined by measuring the uptake of [14C]methylammonium ion (14CH3NH3+) into the intact cells. 14CH3NH3+ uptake was detected in cells grown in medium containing glutamate as the sole nitrogen source, but not in those grown in medium containing NH4Cl instead of glutamate. Vibrio ABE-1 did not utilize CH3NH3+ as a carbon or nitrogen source. NH4Cl and nonradiolabeled CH3NH3+ completely inhibited 14CH3NH3+ uptake. These results indicate that 14CH3NH3+ uptake in this bacterium is mediated via an NH4+ transport system and not by a specific carrier for CH3NH3+. The respiratory substrate succinate was required to drive 14CH3NH3+ uptake and the uptake was completely inhibited by KCN, indicating that the uptake was energy dependent. The electrochemical potentials of H+ and/or Na+ across membranes were suggested to be the driving forces for the transport system because the ionophores carbonylcyanide m-chlorophenylhydrazone and monensin strongly inhibited uptake activities at pH 6.5 and 8.5, respectively. Furthermore, KCl activated 14CH3NH3+ uptake. The 14CH3NH3+ uptake activity of Vibrio ABE-1 was markedly high at temperatures between 0° and 15°C, and the apparent Km value for CH3NH3+ of the uptake did not change significantly over the temperature range from 0° to 25°C. Thus, the NH4+ transport system of this bacterium was highly active at low temperatures.