Functional analysis of Arabidopsis thaliana isoforms of the Mg-chelatase CHLI subunit

The first step of chlorophyll biosynthesis is catalyzed by a Mg-chelatase composed of the subunits CHLI, CHLD and CHLH. Mg-chelatase requires ATP hydrolysis that can be attributed to CHLI. Arabidopsis has two CHLI isoforms, CHLI1 and CHLI2, that have similar expression profiles, but it has been suggested that CHLI2 has limited function in the Mg-chelatase complex. Recently, we showed that Arabidopsis CHLI1 is an ATPase and a target of chloroplast thioredoxin. Here, we demonstrate that CHLI2 also has ATPase activity but with a lower Vmax and higher Km ATP than CHLI1. We confirmed the thioredoxin-dependent reduction of a disulfide bond in CHLI2 and thiol-modulation of its ATPase activity. We then examined the physiological contribution of CHLI2 using a chli2 T-DNA knockout line. Although visible phenotype of homozygous chli2 mutants was almost comparable to wild type, the mutant accumulated significantly less chlorophyll. Furthermore, cs/cs; chli2/chli2 double mutants were almost albino. There were three phenotypes among progenies segregated from the cs/cs; CHLI2/chli2 parent: cs-like pale green, yellow, and almost albino were obtained in the approximate ratio of 1:2:0.7. PCR analysis confirmed that the chli2 mutation is semidominant on a homozygous cs background. These results reveal that although CHLI2 plays a limited role in chlorophyll biosynthesis, this subunit certainly contributes to the assembly of the Mg-chelatase complex.     

Mutant screen distinguishes between residues necessary for light-signal perception and signal transfer by phytochrome B

The phytochromes (phyA to phyE) are a major plant photoreceptor family that regulate a diversity of developmental processes in response to light. The N-terminal 651-amino acid domain of phyB (N651), which binds an open tetrapyrrole chromophore, acts to perceive and transduce regulatory light signals in the cell nucleus. The N651 domain comprises several subdomains: the N-terminal extension, the Per/Arnt/Sim (PAS)-like subdomain (PLD), the cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) subdomain, and the phytochrome (PHY) subdomain. To define functional roles for these subdomains, we mutagenized an Arabidopsis thaliana line expressing N651 fused in tandem to green fluorescent protein, beta-glucuronidase, and a nuclear localization signal. A large-scale screen for long hypocotyl mutants identified 14 novel intragenic missense mutations in the N651 moiety. These new mutations, along with eight previously identified mutations, were distributed throughout N651, indicating that each subdomain has an important function. In vitro analysis of the spectral properties of these mutants enabled them to be classified into two principal classes: light-signal perception mutants (those with defective spectral activity), and signaling mutants (those normal in light perception but defective in intracellular signal transfer). Most spectral mutants were found in the GAF and PHY subdomains. On the other hand, the signaling mutants tend to be located in the N-terminal extension and PLD. These observations indicate that the N-terminal extension and PLD are mainly involved in signal transfer, but that the C-terminal GAF and PHY subdomains are responsible for light perception. Among the signaling mutants, R110Q, G111D, G112D, and R325K were particularly interesting. Alignment with the recently described three-dimensional structure of the PAS-GAF domain of a bacterial phytochrome suggests that these four mutations reside in the vicinity of the phytochrome light-sensing knot.     

A new -2-haloacid dehalogenase acting on 2-haloacid amides: purification, characterization, and mechanism

-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of - and -2-haloalkanoic acids to produce the corresponding - and -2-hydroxyalkanoic acids, respectively. We have constructed an overproduction system for -2-haloacid dehalogenase from Pseudomonas putida PP3 ( -DEX 312) and purified the enzyme to analyze the reaction mechanism. When a single turnover reaction of -DEX 312 was carried out in H₂¹⁸O by use of a large excess of the enzyme with - or -2-chloropropionate as a substrate, the lactate produced was labeled with ¹⁸O. This indicates that the solvent water molecule directly attacked the substrate and that its oxygen atom was incorporated into the product. This reaction mechanism contrasts with that of -2-haloacid dehalogenase, which has an active-site carboxylate group that attacks the substrate to displace the halogen atom. -DEX 312 resembles -2-haloacid dehalogenase from Pseudomonas sp. 113 ( -DEX 113) in that the reaction proceeds with a direct attack of a water molecule on the substrate. However, -DEX 312 is markedly different from -DEX 113 in its substrate specificity. We found that -DEX 312 catalyzes the hydrolytic dehalogenation of 2-chloropropionamide and 2-bromopropionamide, which do not serve as substrates for -DEX 113. -DEX 312 is the first enzyme that catalyzes the dehalogenation of 2-haloacid amides.     

Purification, characterization, and gene cloning of a novel fluoroacetate dehalogenase from Burkholderia sp. FA1

Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of the highly stable carbon–fluorine bond in an aliphatic compound. The bacterial isolate FA1, which was identified as Burkholderia, grew on fluoroacetate as the sole carbon source to produce fluoroacetate dehalogenase (FAc-DEX FA1). The enzyme was purified to homogeneity and characterized. The molecular weights were estimated to be 79,000 and 34,000 by gel filtration and SDS-polyacrylamide gel electrophoresis (PAGE), respectively, suggesting that the enzyme is a dimer. The purified enzyme was specific to haloacetates, and fluoroacetate was the best substrate. The activities toward chloroacetate and bromoacetate were less than 5% of the activity toward fluoroacetate. The K_m and V_max values for the hydrolysis of fluoroacetate were 5.1 mM and 11 μmol per minute milligram, respectively. The gene coding for the enzyme was isolated, and the nucleotide sequence was determined. The open reading frame consisted of 912 nucleotides, corresponding to 304 amino acid residues. Although FAc-DEX FA1 showed high sequence similarity to fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX H1) (61% identity), the substrate specificity of FAc-DEX FA1 was significantly different from that of FAc-DEX H1: FAc-DEX FA1 was more specific to fluoroacetate than FAc-DEX H1.     

Stereoselective reduction of keto esters: thermophilic bacteria and microalgae as new biocatalysts

This review covers the possibility of aerobic thermophilic bacteria (Bacillus strains and thermophilic actinomycetes) and microalgae (Chlorella strains and marine algae) as new biocatalysts for the stereoselective reduction of - and β-keto esters. The mechanistic interpretation of the reduction by a thermophilic actinomycete is also delineated.     

Conversion of the catalytic specificity of alanine racemase to a -amino acid aminotransferase activity by a double active-site mutation

Alanine racemase depending on pyridoxal 5′-phosphate catalyzes the interconversion between - and -alanine. The enzyme from Bacillus stearothermophilus catalyzes the transamination as a side reaction with both substrates once per 3×10⁷ times of the racemization. In this work, we studied the effects of the mutation of Arg219, and that of Arg219 and Tyr265 on the catalysis of Bacillus alanine racemase. Arg219 interacting with pyridinium nitrogen of the cofactor is conserved in all alanine racemases. The corresponding residue of aminotransferases is an acidic residue, such as glutamate or aspartate. Mutation of Arg219 to a glutamyl residue resulted in a 5.4-fold increase in the forward half transamination activity with -alanine and a 10³-fold decrease in the racemase activity. The double mutation, Arg219→Glu and Tyr265→Ala, completely abolished the racemase activity and increased the forward half transaminase activity 6.6-fold. Arg219 is one of the structural determinants of the catalytic specificity of the alanine racemase.     

-Amino acid aminotransferase: fragmentation at a flexible loop is an efficient method to generate mutant enzymes with new substrate specificities and elevated activities

-Amino acid aminotransferase ( -AAT) (EC 2.6.1.21) catalyzes the interconversion between various -amino acids and -keto acids. A subunit of the homodimeric enzyme from a thermophile, Bacillus sp. YM-1, consists of two distinct structural domains connected by one loop. We previously constructed an active fragmentary enzyme whose backbone was cut at the interdomain loop [J. Biochem. 124 (1998) 905]. In this work, we constructed 13 fragmentary -amino acid aminotransferase genes by inserting a termination codon, an SD sequence, and an initiation codon into the specific positions of the gene corresponding to various loop regions and expressed in Escherichia coli cells. We have obtained six genes producing active fragmentary enzymes, one producing an inactive fragmentary enzyme, four producing only large peptide fragment, and another two that gave no products. The six active fragmentary enzymes purified to near-homogeneity showed various substrate specificities and thermostabilities distinct from each other and also from the wild-type enzyme: two exhibited higher catalytic activity towards -alanine, the most efficient substrate, than the wild-type enzyme. These results suggest that cleavage at a loop region is an efficient method for the alteration of enzyme properties.     

Ethylene and salicylic acid control glutathione biosynthesis in ozone‐exposed Arabidopsis thaliana

Ozone produces reactive oxygen species and induces the synthesis of phytohormones, including ethylene and salicylic acid. These phytohormones act as signal molecules that enhance cell death in response to ozone exposure. However, some studies have shown that ethylene and salicylic acid can instead decrease the magnitude of ozone‐induced cell death. Therefore, we studied the defensive roles of ethylene and salicylic acid against ozone. Unlike the wild‐type, Col‐0, Arabidopsis mutants deficient in ethylene signaling (ein2) or salicylic acid biosynthesis (sid2) generated high levels of superoxide and exhibited visible leaf injury, indicating that ethylene and salicylic acid can reduce ozone damage. Macroarray analysis suggested that the ethylene and salicylic acid defects influenced glutathione (GSH) metabolism. Increases in the reduced form of GSH occurred in Col‐0 6 h after ozone exposure, but little GSH was detected in ein2 and sid2 mutants, suggesting that GSH levels were affected by ethylene or salicylic acid signaling. We performed gene expression analysis by real‐time polymerase chain reaction using genes involved in GSH metabolism. Induction of γ‐glutamylcysteine synthetase (GSH1), glutathione synthetase (GSH2), and glutathione reductase 1 (GR1) expression occurred normally in Col‐0, but at much lower levels in ein2 and sid2. Enzymatic activities of GSH1 and GSH2 in ein2 and sid2 were significantly lower than in Col‐0. Moreover, ozone‐induced leaf damage observed in ein2 and sid2 was mitigated by artificial elevation of GSH content. Our results suggest that ethylene and salicylic acid protect against ozone‐induced leaf injury by increasing de novo biosynthesis of GSH.     

Localization of insulinoma associated protein 2, IA-2 in mouse neuroendocrine tissues using two novel monoclonal antibodies

AimsInsulinoma-associated protein 2 (IA-2) is a member of the protein tyrosine phosphatase family that is localized on the insulin granule membrane. IA-2 is also well known as one of the major autoantigens in Type 1 diabetes mellitus. IA-2 gene deficient mice were recently established and showed abnormalities in insulin secretion. Thus, detailed localization of IA-2 was studied using wild-type and IA-2 gene deficient mice.Main methodsTo localize IA-2 expression in mouse neuroendocrine tissues, monoclonal antibodies were generated against IA-2 and western blot and immunohistochemical analyses were carried out in IA-2^+/+ mice. IA-2^?/? mice served as a negative control.Key findingsWestern blot analysis revealed that the 65 kDa form of IA-2 was observed in the cerebrum, cerebellum, medulla oblongata, pancreas, adrenal gland, pituitary gland, muscular layers of the stomach, small intestine, and colon. By immunohistochemical analysis, IA-2 was produced in endocrine cells in pancreatic islets, adrenal medullary cells, thyroid C-cells, Kulchitsky cells, and anterior, intermediate, and posterior pituitary cells. In addition, IA-2 was found in somatostatin-producing D-cells and other small populations of cells were scattered in the gastric corpus. IA-2 expression in neurites was confirmed by the immunostaining of IA-2 using primary cultured neurons from the small intestine and nerve growth factor (NGF)-differentiated PC12 cells.SignificanceThe IA-2 distribution in peripheral neurons appeared more intensely in neurites rather than in the cell bodies.     

Systematic phenome analysis of Escherichia coli multiple‐knockout mutants reveals hidden reactions in central carbon metabolism

Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple‐knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of sedoheptulose‐7‐phosphate to erythrose‐4‐phosphate and dihydroxyacetone phosphate were observed in transaldolase‐deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of sedoheptulose‐7‐phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP‐dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks.