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1.  Is d-aspartate produced by glutamic-oxaloacetic transaminase-1 like 1 (Got1l1): a putative aspartate racemase? 
Amino Acids  2014;47:79-86.
d-Aspartate is an endogenous free amino acid in the brain, endocrine tissues, and exocrine tissues in mammals, and it plays several physiological roles. In the testis, d-aspartate is detected in elongate spermatids, Leydig cells, and Sertoli cells, and implicated in the synthesis and release of testosterone. In the hippocampus, d-aspartate strongly enhances N-methyl-d-aspartate receptor-dependent long-term potentiation and is involved in learning and memory. The existence of aspartate racemase, a candidate enzyme for d-aspartate production, has been suggested. Recently, mouse glutamic-oxaloacetic transaminase 1-like 1 (Got1l1) has been reported to synthesize substantially d-aspartate from l-aspartate and to be involved in adult neurogenesis. In this study, we investigated the function of Got1l1 in vivo by generating and analyzing Got1l1 knockout (KO) mice. We also examined the enzymatic activity of recombinant Got1l1 in vitro. We found that Got1l1 mRNA is highly expressed in the testis, but it is not detected in the brain and submandibular gland, where d-aspartate is abundant. The d-aspartate contents of wild-type and Got1l1 KO mice were not significantly different in the testis and hippocampus. The recombinant Got1l1 expressed in mammalian cells showed l-aspartate aminotransferase activity, but lacked aspartate racemase activity. These findings suggest that Got1l1 is not the major aspartate racemase and there might be an as yet unknown d-aspartate-synthesizing enzyme.
PMCID: PMC4282708  PMID: 25287256
Glutamic-oxaloacetic transaminase-1 like 1; d-Aspartate; Knockout mice; Testis; Hippocampus; Recombinant protein expression
2.  Geranylgeranyl Reductase and Ferredoxin from Methanosarcina acetivorans Are Required for the Synthesis of Fully Reduced Archaeal Membrane Lipid in Escherichia coli Cells 
Journal of Bacteriology  2014;196(2):417-423.
Archaea produce membrane lipids that typically possess fully saturated isoprenoid hydrocarbon chains attached to the glycerol moiety via ether bonds. They are functionally similar to, but structurally and biosynthetically distinct from, the fatty acid-based membrane lipids of bacteria and eukaryotes. It is believed that the characteristic lipid structure helps archaea survive under severe conditions such as extremely low or high pH, high salt concentrations, and/or high temperatures. We detail here the first successful production of an intact archaeal membrane lipid, which has fully saturated isoprenoid chains, in bacterial cells. The introduction of six phospholipid biosynthetic genes from a methanogenic archaeon, Methanosarcina acetivorans, in Escherichia coli enabled the host bacterium to synthesize the archaeal lipid, i.e., diphytanylglyceryl phosphoglycerol, while a glycerol modification of the phosphate group was probably catalyzed by endogenous E. coli enzymes. Reduction of the isoprenoid chains occurred only when archaeal ferredoxin was expressed with geranylgeranyl reductase, suggesting the role of ferredoxin as a specific electron donor for the reductase. This report is the first identification of a physiological reducer for archaeal geranylgeranyl reductase. On the other hand, geranylgeranyl reductase from the thermoacidophilic archaeon Sulfolobus acidocaldarius could, by itself, replace both its orthologue and ferredoxin from M. acetivorans, which indicated that an endogenous redox system of E. coli reduced the enzyme.
PMCID: PMC3911245  PMID: 24214941
3.  Conserved Pyridoxal Protein That Regulates Ile and Val Metabolism 
Journal of Bacteriology  2013;195(24):5439-5449.
Escherichia coli YggS is a member of the highly conserved uncharacterized protein family that binds pyridoxal 5′-phosphate (PLP). To assist with the functional assignment of the YggS family, in vivo and in vitro analyses were performed using a yggS-deficient E. coli strain (ΔyggS) and a purified form of YggS, respectively. In the stationary phase, the ΔyggS strain exhibited a completely different intracellular pool of amino acids and produced a significant amount of l-Val in the culture medium. The log-phase ΔyggS strain accumulated 2-ketobutyrate, its aminated compound 2-aminobutyrate, and, to a lesser extent, l-Val. It also exhibited a 1.3- to 2.6-fold increase in the levels of Ile and Val metabolic enzymes. The fact that similar phenotypes were induced in wild-type E. coli by the exogenous addition of 2-ketobutyrate and 2-aminobutyrate indicates that the 2 compounds contribute to the ΔyggS phenotypes. We showed that the initial cause of the keto acid imbalance was the reduced availability of coenzyme A (CoA); supplementation with pantothenate, which is a CoA precursor, fully reversed phenotypes conferred by the yggS mutation. The plasmid-borne expression of YggS and orthologs from Bacillus subtilis, Saccharomyces cerevisiae, and humans fully rescued the ΔyggS phenotypes. Expression of a mutant YggS lacking PLP-binding ability, however, did not reverse the ΔyggS phenotypes. These results demonstrate for the first time that YggS controls Ile and Val metabolism by modulating 2-ketobutyrate and CoA availability. Its function depends on PLP, and it is highly conserved in a wide range species, from bacteria to humans.
PMCID: PMC3889608  PMID: 24097949
4.  Substrate-Induced Change in the Quaternary Structure of Type 2 Isopentenyl Diphosphate Isomerase from Sulfolobus shibatae 
Journal of Bacteriology  2012;194(12):3216-3224.
Type 2 isopentenyl diphosphate isomerase catalyzes the interconversion between two active units for isoprenoid biosynthesis, i.e., isopentenyl diphosphate and dimethylallyl diphosphate, in almost all archaea and in some bacteria, including human pathogens. The enzyme is a good target for discovery of antibiotics because it is essential for the organisms that use only the mevalonate pathway to produce the active isoprene units and because humans possess a nonhomologous isozyme, type 1 isopentenyl diphosphate isomerase. However, type 2 enzymes were reportedly inhibited by mechanism-based drugs for the type 1 enzyme due to their surprisingly similar reaction mechanisms. Thus, a different approach is now required to develop new inhibitors specific to the type 2 enzyme. X-ray crystallography and gel filtration chromatography revealed that the enzyme from a thermoacidophilic archaeon, Sulfolobus shibatae, is in the octameric state at a high concentration. Interestingly, a part of the regions that are involved in the substrate binding in the previously reported tetrameric structures is integral to the formation of the tetramer-tetramer interface in the substrate-free octameric structure. Site-directed mutagenesis at such regions resulted in stabilization of the tetramer. Small-angle X-ray scattering, tryptophan fluorescence, and dynamic light scattering analyses showed that substrate binding causes the dissociation of an octamer into tetramers. This property, i.e., incompatibility between octamer formation and substrate binding, might provide clues to develop new specific inhibitors of the archaeal enzyme.
PMCID: PMC3370841  PMID: 22505674
5.  Archaeal Phospholipid Biosynthetic Pathway Reconstructed in Escherichia coli 
Archaea  2012;2012:438931.
A part of the biosynthetic pathway of archaeal membrane lipids, comprised of 4 archaeal enzymes, was reconstructed in the cells of Escherichia coli. The genes of the enzymes were cloned from a mesophilic methanogen, Methanosarcina acetivorans, and the activity of each enzyme was confirmed using recombinant proteins. In vitro radioassay showed that the 4 enzymes are sufficient to synthesize an intermediate of archaeal membrane lipid biosynthesis, that is, 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphate, from precursors that can be produced endogenously in E. coli. Introduction of the 4 genes into E. coli resulted in the production of archaeal-type lipids. Detailed liquid chromatography/electron spray ionization-mass spectrometry analyses showed that they are metabolites from the expected intermediate, that is, 2,3-di-O-geranylgeranyl-sn-glycerol and 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphoglycerol. The metabolic processes, that is, dephosphorylation and glycerol modification, are likely catalyzed by endogenous enzymes of E. coli.
PMCID: PMC3357500  PMID: 22645416
6.  Specific Partial Reduction of Geranylgeranyl Diphosphate by an Enzyme from the Thermoacidophilic Archaeon Sulfolobus acidocaldarius Yields a Reactive Prenyl Donor, Not a Dead-End Product ▿  
Journal of Bacteriology  2008;190(11):3923-3929.
Geranylgeranyl reductase from Sulfolobus acidocaldarius was shown to catalyze the reduction of geranylgeranyl groups in the precursors of archaeal membrane lipids, generally reducing all four double bonds. However, when geranylgeranyl diphosphate was subjected to the reductase reaction, only three of the four double bonds were reduced. Mass spectrometry and acid hydrolysis indicated that the allylic double bond was preserved in the partially reduced product derived from geranylgeranyl diphosphate. Thus, the reaction product was shown to be phytyl diphosphate, which is a substrate for archaeal prenyltransferases, unlike the completely reduced compound phytanyl diphosphate.
PMCID: PMC2395040  PMID: 18375567
7.  A Novel Lipolytic Enzyme, YcsK (LipC), Located in the Spore Coat of Bacillus subtilis, Is Involved in Spore Germination▿  
Journal of Bacteriology  2007;189(6):2369-2375.
The predicted amino acid sequence of Bacillus subtilis ycsK exhibits similarity to the GDSL family of lipolytic enzymes. Northern blot analysis showed that ycsK mRNA was first detected from 4 h after the onset of sporulation and that transcription of ycsK was dependent on SigK and GerE. The fluorescence of the YcsK-green fluorescent protein fusion protein produced in sporulating cells was detectable in the mother cell but not in the forespore compartment under fluorescence microscopy, and the fusion protein was localized around the developing spores dependent on CotE, SafA, and SpoVID. Inactivation of the ycsK gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, lysozyme, or chloroform. The germination of ycsK spores in a mixture of l-asparagine, d-glucose, d-fructose, and potassium chloride and LB medium was also the same as that of wild-type spores, but the mutant spores were defective in l-alanine-stimulated germination. In addition, zymogram analysis demonstrated that the YcsK protein heterologously expressed in Escherichia coli showed lipolytic activity. We therefore propose that ycsK should be renamed lipC. This is the first study of a bacterial spore germination-related lipase.
PMCID: PMC1899377  PMID: 17220230
8.  Functional Characterization of Alanine Racemase from Schizosaccharomyces pombe: a Eucaryotic Counterpart to Bacterial Alanine Racemase 
Journal of Bacteriology  2001;183(7):2226-2233.
Schizosaccharomyces pombe has an open reading frame, which we named alr1+, encoding a putative protein similar to bacterial alanine racemase. We cloned the alr1+ gene in Escherichia coli and purified the gene product (Alr1p), with an Mr of 41,590, to homogeneity. Alr1p contains pyridoxal 5′-phosphate as a coenzyme and catalyzes the racemization of alanine with apparent Km and Vmax values as follows: for l-alanine, 5.0 mM and 670 μmol/min/mg, respectively, and for d-alanine, 2.4 mM and 350 μmol/min/mg, respectively. The enzyme is almost specific to alanine, but l-serine and l-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that of l-alanine, respectively. S. pombe uses d-alanine as a sole nitrogen source, but deletion of the alr1+ gene resulted in retarded growth on the same medium. This indicates that S. pombe has catabolic pathways for both enantiomers of alanine and that the pathway for l-alanine coupled with racemization plays a major role in the catabolism of d-alanine. Saccharomyces cerevisiae differs markedly from S. pombe: S. cerevisiae uses l-alanine but not d-alanine as a sole nitrogen source. Moreover, d-alanine is toxic to S. cerevisiae. However, heterologous expression of the alr1+ gene enabled S. cerevisiae to grow efficiently on d-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of d-alanine.
PMCID: PMC95128  PMID: 11244061
9.  Cold-Active Serine Alkaline Protease from the Psychrotrophic Bacterium Shewanella Strain Ac10: Gene Cloning and Enzyme Purification and Characterization 
The gene encoding serine alkaline protease (SapSh) of the psychrotrophic bacterium Shewanella strain Ac10 was cloned in Escherichia coli. The amino acid sequence deduced from the 2,442-bp nucleotide sequence revealed that the protein was 814 amino acids long and had an estimated molecular weight of 85,113. SapSh exhibited sequence similarities with members of the subtilisin family of proteases, and there was a high level of conservation in the regions around a putative catalytic triad consisting of Asp-30, His-65, and Ser-369. The amino acid sequence contained the following regions which were assigned on the basis of homology to previously described sequences: a signal peptide (26 residues), a propeptide (117 residues), and an extension up to the C terminus (about 250 residues). Another feature of SapSh is the fact that the space between His-65 and Ser-369 is approximately 150 residues longer than the corresponding spaces in other proteases belonging to the subtilisin family. SapSh was purified to homogeneity from the culture supernatant of E. coli recombinant cells by affinity chromatography with a bacitracin-Sepharose column. The recombinant SapSh (rSapSh) was found to have a molecular weight of about 44,000 and to be highly active in the alkaline region (optimum pH, around 9.0) when azocasein and synthetic peptides were used as substrates. rSapSh was characterized by its high levels of activity at low temperatures; it was five times more active than subtilisin Carlsberg at temperatures ranging from 5 to 15°C. The activation energy for hydrolysis of azocasein by rSapSh was much lower than the activation energy for hydrolysis of azocasein by the subtilisin. However, rSapSh was far less stable than the subtilisin.
PMCID: PMC91069  PMID: 9925590

Results 1-9 (9)