The structural genes of two homologous enzymes, 6-aminohexanoate-dimer hydrolase (EII ; nylB) and its evolutionally related protein EII' (nylB') of Flavobacterium sp. K172 have an open reading frame encoding a peptide of 392 amino acids, of which 47 are different, and conserved restriction sites. The specific activity of EII towards 6-aminohexanoate dimer is about 1000-fold that of EII'. Construction of various hybrid genes obtained by exchanging fragments flanked by conserved restriction sites of the two genes demonstrated that two amino acid replacements in the EII' enzyme, i.e. Glyl8l -+ A s p (EII type) and His266 -+ Asn (ELI type), enhanced the activity toward 6-aminohexanoate dimer 1000-fold.Enzymes responsible for the degradation of man-made compounds are interesting in terms of evolution. We have shown that two enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (EI) [l] and 6-aminohexanoate-dimer hydrolase (EII) 121 are responsible for Flavobacterium sp. K172 degrading 6-aminohexanoate cyclic dimer, a by-product of nylon manufacture. The EI gene (nylA) and EII gene (nylB) are encoded on pOAD2, one of the three plasmids harbored in Flavobacterium sp. K172 131. This plasmid contains two repeated sequences, RS-I and RS-11; one of the two RS-I1 regions, RS-IIA, contains the nylB gene, while the other, RS-IIB, contains the homologous EII' gene (nylB') [4]. Both genes have the same size open reading frame encoding a peptide of 392 amino acids, of which 47 are different [5] (Fig. 1). Despite their sequence similarity, the specific activity of EII' toward 6-aminohexanoate dimer is 0.1 -1 % of that of EII [5]. This raised the question of which amino acid alterations out of the 47 amino acid alterations were essential for increasing the hydrolase activity. We showed that four amino acid alterations included in the BglII -SalI fragment (residues 162 -257 of the amino acid sequences) are necessary for increasing the activity, and that this effect is enhanced by 15 amino acid alterations in the SaZI -BamHI region (residues 258 -380)In this paper, we identified minimum amino acid alter-[61.ations which affect the catalytic activity. MATERIALS AND METHODS Microorganisms, plasmids and cultivationHybrid plasmids containing the nylB gene, pNL212d 10 [4] and pHK4 [6], and a hybrid plasmid containing the nylB' gene, pHKl [6], had been constructed previously. pNL212d 10-1, which lacked a 70-bp fragment including the nylB gene, was constructed from pNL212d 10 by BurnHI digestion followed by ligation. Plasmids producing the EII-EII' hybrid enzymes, pHK2, pHK5, pHK7 and pHK8 [6], had been constructed previously. The vector pUC12 [7] was previously reported. Escherichia coli C600rK-mK-(thr-1 leuB6 thi-1 supE44 lacy1 tonA2I hsdM hsdR) [8] was host for pHK plasmids. E. coli strains were grown at 37 "C on Luria-Bertani (LB) medium [9] to a density of between 2 x lo9 cells/ml and 3 x lo9 cells/ ml. When necessary, ampicillin (50 pg/ml) was added to the medium. Enzymes and chemicalsThe restriction endonuclease BssHII was obtained from...
A bacterium, Ochrobactrum anthropi, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversible N-riboside hydrolysis of purine nucleosides, the K m values being 11.8 to 56.3 M. The optimal activity temperature and pH were 50°C and pH 4.5 to 6.5, respectively. Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme. The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with K i and K i values of 0.455 to 11.2 M. Metal ion chelators inhibited activity, and the addition of Zn 2؉ or Co 2؉ restored activity. A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed in Escherichia coli. The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH 2 -terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites. The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases.Recently, nucleosides and a variety of chemically synthesized nucleoside analogs have attracted a great deal of interest, as they have antibiotic, antiviral, and antitumoral effects (9). In light of this trend, we conducted studies on the microbial metabolism of nucleosides (5). In this study, we found that a bacterium, Ochrobactrum anthropi, shows a high level of activity in the N-riboside cleavage of purine nucleosides. This reaction is important in the decomposition of purine nucleosides in foodstuffs which cause hyperuricemia, an increasingly common disease in adults (3).The enzymatic N-riboside cleavage of nucleosides is a common reaction in various organisms (1,20). This reaction seems to participate in a salvage or assimilation pathway for nucleosides. Two kinds of enzymes, nucleoside phosphorylases (EC 2.4.2.-) and nucleosidases (nucleoside hydrolase; EC 3.2.2.-), are known to catalyze this reaction. Nucleoside phosphorylases catalyze the phosphorolytic cleavage of nucleosides and show ribosyl transferase activity (7). These enzymes play roles mainly in the salvage pathway. Nucleoside phosphorylases have been well studied. In addition, they have been purified from various sources and used as catalysts for the synthesis of nucleoside analogs through base exchange reactions (7, 21). In contrast, nucleosidases catalyze the irreversible hydrolysis of nucleosides and participate mainly in the assimilation pathway.There have been few studies on microbial nucleosidases acting on purine and pyrimidine nucleosides and no reports of homogenously purified bacterial purine and pyrimidine nucleosidases (8,18,19).In this study, we report the...
The maltose transporter gene is situated at the MAL locus, which consists of genes for a transporter, maltase, and transcriptional activator. Five unlinked MAL loci (MAL1, MAL2, MAL3, MAL4, and MAL6) constitute a gene family in Saccharomyces cerevisiae. The expression of the maltose transporter is induced by maltose and repressed by glucose. The activity of the maltose transporter is also regulated post-translationally; Mal61p is rapidly internalized from the plasma membrane and degraded by ubiquitin-mediated proteolysis in the presence of glucose. We found that S. cerevisiae strain ATCC20598 harboring MAL21 could grow in maltose supplemented with a non-assimilable glucose analogue, 2-deoxyglucose, whereas strain ATCC96955 harboring MAL61 and strain CB11 with MAL31 and AGT1 could not. These observations implied a Mal21p-specific resistance against glucose-induced degradation. Mal21p found in ATCC20598 has 10 amino acids, including Gly-46 and His-50, that are inconsistent with the corresponding residues in Mal61p. The half-life of Mal21p for glucose-induced degradation was 118 min when expressed using the constitutive TPI1 promoter, which was significantly longer than that of Mal61p (25 min). Studies with mutant cells that are defective in endocytosis or the ubiquitination process indicated that Mal21p was less ubiquitinated than Mal61p, suggesting that Mal21p remains on the plasma membrane because of poor susceptibility to ubiquitination. Mutational studies revealed that both residues Gly-46 and His-50 in Mal21p are essential for the full resistance of maltose transporters against glucose-induced degradation.In yeast, the expression of sugar transporters is strictly regulated at transcriptional, post-transcriptional, translational, and post-translational levels for efficient sugar assimilation and glycolysis flux control. Hexose transporters transfer their substrates by facilitated diffusion, whereas maltose transporters transport maltose via proton symport driven by a proton gradient maintained by ATP consumption. Saccharomyces cerevisiae assimilates glucose preferentially because it does not require energy consumption, whereas the transporters for other sugars are down-regulated when glucose is available.
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