Comparative biochemistry of bacterial N-acyl- -amino acid amidohydrolase

N-acyl- -amino acid amidohydrolases can be classified into three types based on substrate specificity. -aminoacylase has been reported to occur in a very few bacteria such as Pseudomonas, Streptomyces, and Alcaligenes. N-acyl- -aspartate amidohydrolase ( -AAase) has been reported in only Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) while N-acyl- -glutamate amidohydrolase ( -AGase) has been isolated in two stains of Pseudomonas sp. 5f-1 and Alcaligenes A-6. The physiological roles of these enzymes in these microbes are not clear. They are individually characteristic in their substrate specificities, inducer profiles, inhibitors, isoelectric points, metal dependency, and some physicochemical properties. The primary structures of all the three types of N-acyl- -amino acid amidohydrolases from Alcaligenes A-6 were determined from their nucleotide sequences. Comparison of their primary structures revealed high homology (46–56%) between the different enzymes. The three enzymes showed 26–27% sequence homology with -aminoacylases from Bacillus stearothermophilus, porcine, and human. Chemical modification and site-directed mutagenesis identified the histidyl residues essential for catalysis. The Alcaligenes N-acyl- -amino acid amidohydrolases share significant sequence similarities with some members of the urease-related amidohydrolase superfamily proposed by Holm and Sander [L. Holm, C. Sander, Proteins: Structure, Function and Genetics 28 (1997) 72].     

Synthesis and evaluation of N8-acetylspermidine analogues as inhibitors of bacterial acetylpolyamine amidohydrolase

Polyamines are small essential polycations involved in many biological processes. Enzymes of polyamine metabolism have been extensively studied and are attractive drug targets. Nevertheless, the reversible acetylation of polyamines remains poorly understood. Although eukaryotic N⁸-acetylspermidine deacetylase activity has already been detected and studied, the specific enzyme responsible for this activity has not yet been identified. However, a zinc deacetylase from Mycoplana ramosa, acetylpolyamine amidohydrolase (APAH), has been reported to use various acetylpolyamines as substrates. The recently solved crystal structure of this polyamine deacetylase revealed the formation of an ‘L’-shaped active site tunnel at the dimer interface, with ideal dimensions and electrostatic properties for accommodating narrow, flexible, cationic polyamine substrates. Here, we report the design, synthesis, and evaluation of N⁸-acetylspermidine analogues bearing different zinc binding groups as potential inhibitors of APAH. Most of the synthesized compounds exhibit modest potency, with IC₅₀ values in the mid-micromolar range, but compounds bearing hydroxamate or trifluoromethylketone zinc binding groups exhibit enhanced inhibitory potency in the mid-nanomolar range. These inhibitors will enable future explorations of acetylpolyamine function in both prokaryotes and eukaryotes.     

Purification of DNA for bacterial productivity estimates

[methyl-3H]thymidine-labeled DNA from natural populations of aquatic bacteria was completely separated from RNA and protein by hydroxylapatite chromatography. The procedure was validated by monitoring increases in Escherichia coli cell count, A550, DNA concentration, and thymidine incorporation into DNA isolated by the proposed technique. The procedure can be used in the field and does not rely on the use of acid-base hydrolysis or volatile organic solvents.     

Comparison of ribosomal RNA-binding protein "L2" isolated from different bacterial species

Summary A ribosomal protein (L2) which binds to 23S rRNA was isolated from 70S ribosomes of several Bacillacease. It was shown by two-dimensional gel electrophoresis, molecular weight determination, amino acid analysis and immunological methods that this protein is homologous to the E. coli ribosomal protein L2 which also binds to 23S rRNA. In all Bacillaceae this protein L2 remains bound to 23S rRNA after extraction of ribosomal proteins with 4 M urea and 2 M LiCl, in contrast to E. coli. Immunological experiments demonstrated that the protein L2 of B. stearothermophilus undergoes a conformational change when it binds to 23S RNA.Paper No. 69 on Ribosomal proteins. Preceding paper is by Geisser et al., Molec. gen. Genet. 127, 129–145 (173)     

Purification of DNA for bacterial productivity estimates.

[methyl-3H]thymidine-labeled DNA from natural populations of aquatic bacteria was completely separated from RNA and protein by hydroxylapatite chromatography. The procedure was validated by monitoring increases in Escherichia coli cell count, A550, DNA concentration, and thymidine incorporation into DNA isolated by the proposed technique. The procedure can be used in the field and does not rely on the use of acid-base hydrolysis or volatile organic solvents.     

The aerobic dechlorination activities of two bacterial species isolated from a refuse dumpsite in Nigeria

Two bacterial species isolated using enrichment culture techniques from the topsoil of a main refuse dumpsite in Nigeria were assessed for their dehalogenation potentials. The bacterial isolates were identified as belonging to the Bacillus and Pseudomonas genera. Axenic cultures of the isolates utilized monochloroacetic acid (MCA), trichloroacetic acid (TCA), trichloromethane (CHCl₃) and tetrachloromethane (CCl₄) as the sole source of carbon for growth up to a final substrate concentration of 0.1% (w/v). The mean generation times of the isolates in all the growth media ranged significantly (P<0.05) from 2.41 to 10.04 h and were generally higher than that observed in glucose medium (1.46–1.51 h). The numbers of the chloride atoms in the different organochlorides were negatively correlated with the ability of the organisms to degrade the compounds. Dehalogenase specific activities of the cell-mediated cultures ranged from 0.1 to 0.96 μg ml^–1 chloride release (mg protein)^–1 h^–1 and were significantly (P <0.05) higher than that of the cell-free extract [0.09–0.8 μg ml^–1 chloride release (mg protein)^–1 h^–1]. The optimal pH of the dehalogenase activity was found to be 8.0, and the optimal temperature was between 30 and 35 °C. Electronic Publication     

Different temperature dependencies of the charge recombination reaction in photoreaction centers isolated from different bacterial species

We compared the temperature dependency of the rate of the charge recombination reaction in photoreaction centers isolated from Ectothiorhodospira sp. and from Rhodospirillum rubrum G9. We also examined the temperature dependency of the bandwidth and peak wavelength of their far-red absorption band. In both preparations, the peak wavelength and the bandwidth vary monotonically with temperature between 80 and 300 K. However, the rate of the charge recombination reaction has a quite different temperature dependency. In the preparation from R. rubrum, the reaction is accelerated 5-fold in a typical sigmoidal fashion as the temperature is lowered from 300 to 80 K. In the preparation from Ectothiorhodospira sp., the reaction is accelerated monotonically only about 1.5-fold in the same temperature range. At temperatures below 100 K, the rates are similar in the two preparations. We interpret the temperature dependency of the charge recombination reaction in terms of an activationless electron-transfer model formulated by Jortner (Jortner, J. (1980) Biochim. Biophys. Acta 394, 193–230). The minimal model provides a good fit for the temperature dependency of charge recombination in the preparation from Ectothiorhodospira sp. However, to fit the temperature dependency of the R. rubrum preparation with the same model, we must further postulate that the electronic coupling factor varies with temperature in this preparation. We find that, in both preparations, the temperature dependency of the far-red absorption bandwidth is consistent with the assumption that similar vibrational modes are involved in electron transfer and in electronic excitation.     

N-氨甲酰基-D-氨基酸酰胺水解酶的固定化工艺

以TJS环氧基树脂作为载体对N-氨甲酰基-D-氨基酸酰胺水解酶进行固定化,最佳工艺条件为：1g树脂载体大约对应133U酶液,蛋白质量浓度0.35mg/mL,固定时间15h,温度28℃,pH7.5,固定化酶活达到58.5U。蛋白固定率可达97.4%,酶活回收率达到49.3%,得到的固定化酶使用半衰期达到26批。     

Identification of nicotinamide mononucleotide deamidase of the bacterial pyridine nucleotide cycle reveals a novel broadly conserved amidohydrolase family

The pyridine nucleotide cycle is a network of salvage and recycling routes maintaining homeostasis of NAD(P) cofactor pool in the cell. Nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.42), one of the key enzymes of the bacterial pyridine nucleotide cycle, was originally described in Enterobacteria, but the corresponding gene eluded identification for over 30 years. A genomics-based reconstruction of NAD metabolism across hundreds of bacterial species suggested that NMN deamidase reaction is the only possible way of nicotinamide salvage in the marine bacterium Shewanella oneidensis. This prediction was verified via purification of native NMN deamidase from S. oneidensis followed by the identification of the respective gene, termed pncC. Enzymatic characterization of the PncC protein, as well as phenotype analysis of deletion mutants, confirmed its proposed biochemical and physiological function in S. oneidensis. Of the three PncC homologs present in Escherichia coli, NMN deamidase activity was confirmed only for the recombinant purified product of the ygaD gene. A comparative analysis at the level of sequence and three-dimensional structure, which is available for one of the PncC family member, shows no homology with any previously described amidohydrolases. Multiple alignment analysis of functional and nonfunctional PncC homologs, together with NMN docking experiments, allowed us to tentatively identify the active site area and conserved residues therein. An observed broad phylogenomic distribution of predicted functional PncCs in the bacterial kingdom is consistent with a possible role in detoxification of NMN, resulting from NAD utilization by DNA ligase.     

On enhancing productivity of bioethanol with multiple species

The present work is initiated to investigate whether a defined culture comprising a mixture of three yeast species, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia stipitis can ferment a mixture of sugars to produce bioethanol at rates higher than those achieved by pure cultures of the same. For this purpose, we develop models of single species based on the hybrid cybernetic model framework, and simulate fermentations in the mixed culture by combining individual models. An underlying assumption is that the behavior of each species is determined only by the common environment independently of the presence and metabolism of other species. Model performance is thoroughly assessed using the experimental data available in the literature. The dynamic behavior of mixed cultures in mixed culture experiments are accurately predicted by the model reflecting faithfully the simultaneous/sequential uptake patterns of mixed substrates. This model is then used to investigate performance of various possible reactor configurations. With the foregoing species of organisms, mixed culture itself does not lead to a significant increase of bioethanol productivity. Rather, the model shows that substantial improvement is acquired by sequential use of different, properly chosen organisms during fermentation. Thus, the successive use of K. marxianus and P. stipitis is shown to increase bioethanol productivity up to about 58% in comparison to fermentation by single species alone.     

Properties of rat liver N-acylethanolamine amidohydrolase

Rat liver microsomes and mitochondria contain an amidohydrolase which catalyzes the hydrolysis of N-acylethanolamine to ethanolamine and fatty acid. The enzyme is active over a wide range of pH, does not require divalent cations, and is inhibited by sulfhydryl-reactive agents. The detergents Triton X-100, sodium cholate, and sodium dodecyl sulfate are also inhibitory, but sodium taurodeoxycholate has little effect and was therefore used to solubilize the enzyme. The solubilized enzyme exhibits high substrate specificity for long-chain amides of ethanolamine. Amides of propanolamine or higher homologs are hydrolyzed at a drastically slower rate, and isomers prepared from long-chain amine and short-chain hydroxy acid are neither substrates nor inhibitors of the enzyme. Neither ceramide (N-acylsphingosine) nor N,O-diacylethanolamine is hydrolyzed. Both particulate and soluble enzyme preparations also catalyze the synthesis of N-acylethanolamine from ethanolamine and fatty acid, probably by the amidohydrolase acting in reverse.     

Cloning and comparison of the DNA encoding ammelide aminohydrolase and cyanuric acid amidohydrolase from three s-triazine-degrading bacterial strains.

DNA encoding the catabolism of the s-triazines ammelide and cyanuric acid was cloned from Pseudomonas sp. strain NRRLB-12228 and Klebsiella pneumoniae 99 with, as a probe, a 4.6-kb PstI fragment from a third strain, Pseudomonas sp. strain NRRLB-12227, which also encodes these activities. In strains NRRLB-12228 and 99 the ammelide aminohydrolase (trzC) and cyanuric acid amidohydrolase (trzD) genes are located on identical 4.6-kb PstI fragments which are part of a 12.4-kb DNA segment present in both strains. Strain NRRLB-12227 also carries this 12.4-kb DNA segment, except that a DNA segment of 0.8 to 1.85 kb encoding a third enzyme, ammeline aminohydrolase (trzB), has been inserted next to the ammelide aminohydrolase gene with the accompanying deletion of 1.1 to 2.15 kb of DNA. In addition, the s-triazine catabolic genes are flanked in strain NRRLB-12227 by apparently identical 2.2-kb segments that are not present in the other two strains and that seem to cause rearrangements in adjacent DNA.     

Metal-ion susceptibility of oral bacterial species

Aims: This study aimed to evaluate the effect of lead (Pb) on growth of bacterial species related to dental diseases in vitro. Methods and Results: The effects of lead acetate on representative species of the oral flora were examined at 0·1–10 mmol l^?1 and compared with the effect of silver nitrate and ferrous sulfate. The minimal inhibitory concentration of lead acetate was between 0·15 and 5 mmol l^?1 for the bacterial strains tested. The minimal bactericidal concentration of lead acetate for most oral species was detected in the range of 5–10 mmol l^?1. Silver nitrate at a concentration of 1·25 mmol l^?1 was sufficient to exhibit antibacterial activity against almost all bacteria tested. Ferrous sulfate had the lowest effect. Conclusions: The study indicated a general antimicrobial effect of lead on oral bacterial species in the range of 0·15–10 mmol l^?1. The toxicity of silver nitrate was the highest, whereas that of ferrous sulfate was the lowest. Gram‐positive species had a tendency to be less susceptible for metals than Gram‐negatives. Significance and Impact of the Study: The study shows that it is possible that microbiological changes may occur in the dental plaque in children because of toxic exposure of environmental lead.     

Immobilization of thermotolerant N-carbamyl-d-amino acid amidohydrolase

N-Carbamyl-d-amino acid amidohydrolase (DCase), in which amino acid residues were substituted by mutation, followed by the selection based on thermotolerance, showed improved thermostability, by 5° or 10°C, compared to the native DCase. These DCases were immobilized on a macroporous phenol formaldehyde resin, Duolite A-568, and the immobilized thermotolerant enzymes showed higher activity than the immobilized native DCase. From the results of repeated batch reactions, the half-lives of the activities of immobilized thermotolerant DCase, in which Leu was substituted for Pro 203, and immobilized native DCase were 104 and 58 times, respectively. It was revealed that the higher thermotolerance enabled the immobilized enzymes to be more stable in reactions. A reductant, dithiothreitol, also stabilized the enzymes in reactions. Compared with soluble DCase, immobilized DCase was somewhat stable, and its activity was optimum at a lower pH.