Cloning and nucleotide sequence of the aspartase gene of Pseudomonas fluorescens

The aspartase gene (aspA) of Pseudomonas fluorescens was cloned and the nucleotide sequence of the 2,066-base-pair DNA fragment containing the aspA gene was determined. The amino acid sequence of the protein deduced from the nucleotide sequence was confirmed by N- and C-terminal sequence analysis of the purified enzyme protein. The deduced amino acid composition also fitted the previous amino acid analysis results well (Takagi et al. (1984) J. Biochem. 96, 545-552). These results indicate that aspartase of P. fluorescens consists of four identical subunits with a molecular weight of 50,859, composed of 472 amino acid residues. The coding sequence of the gene was preceded by a potential Shine-Dalgarno sequence and by a few promoter-like structures. Following the stop codon there was a structure which is reminiscent of the Escherichia coli rho-independent terminator. The G + C content of the coding sequence was found to be 62.3%. Inspection of the codon usage for the aspA gene revealed as high as 80.0% preference for G or C at the third codon position. The deduced amino acid sequence was 56.3% homologous with that of the enzyme of E. coli W (Takagi et al. (1985) Nucl. Acids Res. 13, 2063-2074). Cys-140 and Cys-430 of the E. coli enzyme, which had been assigned as functionally essential (Ida & Tokushige (1985) J. Biochem. 98, 793-797), were substituted by Ala-140 and Ala-431, respectively, in the P. fluorescens enzyme.     

Effects of temperature and monovalent cations on activity and quaternary structure of tryptophanase

Effects of temperature and monovalent cations on the activity and the quaternary structure of tryptophanase of Escherichia coli were studied. The conversion of the apoenzyme into the active holoenzyme was attained at 30 degrees C in Tris-HCl buffer (pH 8.0) containing pyridoxal-P and K+, while no conversion occurred at 5 degrees C. The active holoenzyme thus formed was stable even at 5 degrees C, as long as the cation was present. When K+ was absent, however, the active enzyme gradually lost the activity upon chilling to 5 degrees C. The HPLC gel filtration analysis of the active holoenzyme and the low temperature-inactivated enzyme species revealed that the tetrameric holoenzyme dissociated into the dimeric apoenzyme concomitant with the low temperature-induced inactivation at 5 degrees C. The results of HPLC experiments together with other available evidence also suggest that the inactive tetrameric holoenzyme was first formed from the dimeric apoenzyme and pyridoxal-P prior to the formation of the active holoenzyme and that the cation promoted the conversion of the inactive holoenzyme into the active holoenzyme rather than being involved in the conversion of the apoenzyme and pyridoxal-P into the holoenzyme. Among various cations tested for the above effects, NH4+ exhibited the largest effect and K+ the second.     

Studies on aspartase VIII. Protease-mediated activation: comparative survey of protease specificity for activation and peptide cleavage

The active species of aspartase from Escherichia coli is further 3-5 fold activated upon limited proteolysis with trypsin releasing carboxy-terminal peptides as reported previously (N. Yumoto, M. Tokushige, and R. Hayashi. Biochim. Biophys. Acta, 616, 319 (1980) ). Survey of the protease specificity for the activation revealed that subtilisin BPN' and several other proteases having far broader substrate specificity than trypsin also activated the enzyme. The results of sequence analyses revealed that subtilisin BPN' cleaved mainly the serylarginine bond near the carboxy-terminal and released an octapeptide, while trypsin cleaved mainly the arginyltyrosine bond which is just next to the subtilisin cleavage site. These results suggest that the protease-mediated activation does not necessarily require a site-specific peptidyl cleavage, but the cleavage of any bond within a certain region centered at arginine, the eighth residue from the carboxy-terminal, is sufficient.     

Identification of lysyl residues at the AMP-binding site of biodegradative threonine deaminase from Escherichia coli.

The biodegradative threonine deaminase from Escherichia coli is activated allosterically by AMP. To identify the residues interacting with the phosphate group of AMP at the binding site, we used the affinity labeling reagent, adenosine diphosphopyridoxal (AP2-PL). In the absence of AMP, the enzyme formed the Schiff base with AP2-PL and Scatchard plot analysis showed a biphasic pattern, the respective Kd values for the high- and low-affinity binding phases being 20 and 110 microM. The former value is comparable to the Kd value of the enzyme for AMP. In the presence of AMP, the Schiff base formation was greatly reduced. Although the maximal activating effect of adenosine diphosphopyridoxine, a non-reactive derivative of AP2-PL, was about 13% of that of AMP, the half-saturation concentration was almost the same. These findings suggest that AP2-PL specifically labeled the lysyl residue(s) at the AMP-binding site of the enzyme. To identify the labeled residue(s), we reduced the modified enzyme with sodium borohydride, then cleaved it with cyanogen bromide and Achromobacter lyticus protease I. Reverse-phase HPLC was used to isolate two labeled peptides from the digest. Their amino acid compositions and sequences showed that Lys-111 and Lys-113 were labeled. We conclude that these two lysyl residues are located around the phosphate group of AMP at the allosteric regulation site of the enzyme.     

Occurrence of thermolabile and regulatory NAD-linked glutamate dehydrogenase in Pseudomonas fluorescens.

NAD-linked glutamate dehydrogeanse [EC 1.4.1.2] was detected together with NADP-linked glutamate dehydrogenase [EC 1.4.1.4] and aspartase [EC 4.3.1.1] in Pseudomonas fluorescens cells. The three enzymes were distinctly separated by DEAE-Sephadex column chromatography. The NAD-linked enzyme was extremely thermolabile and was rapidly inactivated even at temperatures as low as 35--40 degrees C. The combined addition of NAD+ and glutamate, however, effectively stabilized the enzyme. The glutamate saturation profile of the NAD-linked enzyme exhibited cooperativity with a Hill coefficient (n) of 1.4. ATP inhibited the enzyme in an allosteric manner, increasing the n value to 2.2. These results suggest a novel type of metabolic regulation shared by the three enzymes in the biosynthesis and catabolism of amino acids.     

Specific inhibition of aspartase by S-2,3-dicarboxyaziridine

Aspartase of Escherichia coli was inhibited in a competitive manner by S-2,3-dicarboxyazirdine (DCAZ), an antibacterial substance against Aeromonas salmonesida. The inhibition constant (Ki) was 55 microM, which was as low as less than one tenth that of the Km value for the substrate, L-aspartate. In view of the fact that both aspartase and fumarase (J. Greenhut et al. (1985) J. Biol. Chem. 260, 6684-6686) were inhibited by DCAZ in competitive manners, common features of the reaction mechanism of the two enzymes were discussed.     

Redox Buffer Capacity in Water-Rock-Microbe Interaction Systems in Subsurface Environments

An incubation experiment was conducted to estimate redox buffer capacity of “water-rock-microbe” interaction systems in sedimentary rocks. The water chemistry, microbial growth and community structure were analyzed during the incubations. The dissolved oxygen (DO) concentrations and oxidation-reduction potential (ORP) values decreased notably in the presence of active microorganisms, whereas abiotic reactions did not lead to reducing conditions during incubation. The change in microbial community structure suggests that nitrate-reducing and sulfate-reducing bacteria played an important role in reduction of water by using lignite-derived organic matter. These results show that the microbial role is extremely important for the redox buffering capacity in sedimentary rock environments.     

CLAC: a novel Alzheimer amyloid plaque component derived from a transmembrane precursor, CLAC-P/collagen type XXV

We raised monoclonal antibodies against senile plaque (SP) amyloid and obtained a clone 9D2, which labeled amyloid fibrils in SPs and reacted with approximately 50/100 kDa polypeptides in Alzheimer's disease (AD) brains. We purified the 9D2 antigens and cloned a cDNA encoding its precursor, which was a novel type II transmembrane protein specifically expressed in neurons. This precursor harbored three collagen-like Gly-X-Y repeat motifs and was partially homologous to collagen type XIII. Thus, we named the 9D2 antigen as CLAC (collagen-like Alzheimer amyloid plaque component), and its precursor as CLAC-P/collagen type XXV. The extracellular domain of CLAC-P/collagen type XXV was secreted by furin convertase, and the N-terminus of CLAC deposited in AD brains was pyroglutamate modified. Both secreted and membrane-tethered forms of CLAC-P/collagen type XXV specifically bound to fibrillized Abeta, implicating these proteins in beta-amyloidogenesis and neuronal degeneration in AD.