[show abstract][hide abstract] ABSTRACT: The open reading frame yjbR which had been sequenced as a part of the Bacillus subtilis genome project encodes a putative 40.9-kDa protein. The yjbR-coding sequence was slightly similar to those of bacterial sarcosine oxidases and possibly compatible with the tertiary structure of the porcine kidney D-amino acid oxidase. The yjbR gene product was overproduced in Escherichia coli, purified to homogeneity from the recombinant strain, and characterized. This protein effectively catalyzed the oxidation of sarcosine (N-methylglycine), N-ethylglycine and glycine. Lower activities on D-alanine, D-valine, and D-proline were detected although no activities were shown on L-amino acids and other D-amino acids. Since glycine is a product and not a substrate for sarcosine oxidase, this protein is not a type of demethylating enzymes but a novel deaminating oxidase, named glycine oxidase as a common name. Several enzymatic properties of the B. subtilis glycine oxidase were also investigated.
[show abstract][hide abstract] ABSTRACT: The genes encoding creatininase (CrnA; 258 residues) and creatinase (CreA; 411 residues) from Arthrobacter sp. TE1826 were cloned and sequenced. The genes form a cluster with the sarcosine oxidase gene (soxA) and its regulator gene (soxR), which were cloned previously. The deduced amino acid sequences of CrnA and CreA show 35.9% and 63.1% identity, respectively to the corresponding Pseudomonas enzymes. CrnA and CreA were purified from the recombinant strains and characterized. Other open reading frames (creB and crnB), encoding proteins similar to several transporters, were found downstream of creA and crnA, respectively.
MGG - Molecular and General Genetics 04/1998; 257(5):581-6.
[show abstract][hide abstract] ABSTRACT: Serum chloride ion is routinely assayed in clinical laboratories. We have developed a new enzymatic assay for determining the chloride ion concentration. The method involves the use of a mutant sarcosine oxidase, which was created as desired by site-directed mutagenesis and showed chloride-dependent activity. The enzyme which is reactivated by the chloride ion forms hydrogen peroxide from sarcosine. The production of hydrogen peroxide is measured by the 4-amino-antipyrine peroxidase system. The increase of the reaction rate was proportional to the chloride ion concentration. A lag time of the time course was not observed, and the reaction rate for a blank was not detected. Therefore, a rate assay could be adopted. A standard curve of the assay reagent was linear up to 180 mM chloride ion of the sample. The specificity for the bromide ion was 43% of that of the chloride ion, although it was 0% for other ion species. When serum samples were used, within-day coefficient variations (CVs) and day-to-day CVs were below 1.5%. A good correlation with the comparison assay was observed by using 160 samples of normal and abnormal patient sera. This method can easily and reliably be used for the accurate determination of chloride ion concentration in serum or other samples.
[show abstract][hide abstract] ABSTRACT: Sarcosine oxidase from Arthrobacter sp. TE1826 (SoxA) tightly binds with the coenzyme flavin adenine dinucleotide (FAD). The amino-terminal region of this enzyme was recognized as a part of the FAD-binding domain by homology search analysis. Comparison with other structurally well-known flavoproteins suggested that the aspartate residue at position 35 (D-35) and the motif sequence (six residues at positions 12 to 17) were important for the interaction with FAD. Site-directed mutagenesis of each position was performed, and mutant SoxAs were purified and characterized. When D-35 was substituted with glutamate, asparagine, and alanine, it was indicated that the carboxyl group of the side chain interacted with FAD. Changes in the enzyme-bound FAD were also observed from the altered spectral profiles. Thirteen mutant SoxAs were obtained by replacing amino acids in the motif sequence. Most of them showed inhibited or remarkably decreased sarcosine oxidase activity, and their spectral profiles were altered. However, some of them were reactivated by chloride ion. Their spectral profiles also became close to that of wild type in the presence of chloride ion. These results strongly suggest that the inhibition of interaction of enzyme with FAD was caused by the substitution in the motif and that it could be recovered under different conditions.
Applied and Environmental Microbiology 08/1996; 62(7):2405-10. · 3.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Two cysteine residues (C-265 and C-318) in the putative hydrophilic regions of sarcosine oxidase were substituted by using site-directed mutagenesis. Since the mutant with the C-to-S mutation at position 318 (C318S) lost the enzyme activity, C-318 (conserved among sarcosine oxidases) is most likely a part of the active site. C265S, C265A, C265D, and C265R showed nearly the same enzymatic properties as those of the wild type. However, they were much more stable than the wild type in the presence of inhibitors that modified the thiol group. Moreover, they were extremely stable throughout the cultivation of the recombinant strains or even in cell extracts.
Applied and Environmental Microbiology 02/1995; 61(1):367-70. · 3.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Both the neutral protease gene (nprS) and its transcriptional activator gene (nprA) from Bacillus stearothermophilus TELNE were cloned in Bacillus subtilis by using pTB53 as a vector plasmid. The presence of the nprA gene enhanced protease synthesis by about fivefold. The nucleotide sequences of nprS and its flanking regions were determined. nprS was composed of 1,653 base pairs and 551 amino acid residues. A Shine-Dalgarno (SD) sequence was found 9 bases upstream from the translation start site (ATG). The deduced amino acid sequence was very similar to that of another thermostable neutral protease gene, nprM (M. Kubo and T. Imanaka, J. Gen. Microbiol. 134:1883-1892, 1988). the amino acid sequence of the extracellular neutral protease NprS was completely identical to that of NprM. By deletion analysis and substitution of the original promoter with a foreign promoter, it was found that the nprA gene existed upstream of nprS. It was also found that a possible target region (palindromic sequence) of the gene product of nprA existed near the promoter sequence of nprS. The nucleotide sequences of nprA and its flanking regions were determined. The DNA sequence revealed only one large open reading frame, composed of 1,218 base pairs (406 amino acids; molecular weight, 49,097). The SD sequence was found 4 bases upstream from the translation start site (GTG). A possible promoter sequence (TTGAAG for the -35 region and AATTTT for the -10 region) was also found about 20 bases upstream of the SD sequence. The nprA gene was separated from nprS by a typical terminator sequence. By constructing an in-frame fusion between the lacZ gene and the 5' region of the nprA gene, it was demonstrated that the coding region of nprA was indeed translated in vivo. Three palindromic sequences, which were highly homologous with a possible target region by NprA, were also found in the 5' region of the nprA gene. This suggests that eh expression of nprA is autoregulated. From the time course of the production of NprA-LacZ fusion protein, it was indicated that nprA was expressed in late log phase, whereas nprS was expressed in the stationary phase. The NprA protein had consensus regions homologous to the DNA recognition domains of DNA-binding proteins but showed no sequence homology with any other regulatory proteins for protease production. It is inferred that NprA protein binds to the upstream region of nprS promoter and activates transcription of nprS. A new regulatory mechanism by the nprA-nprS genes is discussed.
Journal of Bacteriology 10/1990; 172(9):4861-9. · 3.19 Impact Factor