Thermostable β-Galactosidase from an Extreme Thermophile,<i>Thermus</i>sp. A4: Enzyme Purification and Characterization, and Gene Cloning and Sequencing

Ohtsu, Naomi; Motoshima, Hidemasa; Goto, Kenji; Tsukasaki, Fuji; Matsuzawa, Hiroshi

doi:10.1271/bbb.62.1539

Cited by 62 publications

(38 citation statements)

References 20 publications

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“…(P96567 [7]); and family 42 ␤-galactosidases from Bacillus stearothermophilus (AAA22262 [9]), Carnobacterium piscicola (AAF16519 [2]), Thermus sp. strain A4 (BAA28362 [26]), Thermus sp. strain T2 (CAB07810 [35]), Haloferax alicantei (AAB40123 [10]), Clostridium cellulovorans (AAN05452 [18]), Planococcus sp.…”

Section: Discussionmentioning

confidence: 99%

Characterization of an Exo-β- d -Glucosaminidase Involved in a Novel Chitinolytic Pathway from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

et al. 2003

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We previously clarified that the chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 produces diacetylchitobiose (GlcNAc 2 ) as an end product from chitin. Here we sought to identify enzymes in T. kodakaraensis that were involved in the further degradation of GlcNAc 2 . Through a search of the T. kodakaraensis genome, one candidate gene identified as a putative ␤-glycosyl hydrolase was found in the near vicinity of the chitinase gene. The primary structure of the candidate protein was homologous to the ␤-galactosidases in family 35 of glycosyl hydrolases at the N-terminal region, whereas the central region was homologous to ␤-galactosidases in family 42. The purified protein from recombinant Escherichia coli clearly showed an exo-␤-D-glucosaminidase (GlcNase) activity but not ␤-galactosidase activity. This GlcNase (GlmA Tk ), a homodimer of 90-kDa subunits, exhibited highest activity toward reduced chitobiose at pH 6.0 and 80°C and specifically cleaved the nonreducing terminal glycosidic bond of chitooligosaccharides. The GlcNase activity was also detected in T. kodakaraensis cells, and the expression of GlmA Tk was induced by GlcNAc 2 and chitin, strongly suggesting that GlmA Tk is involved in chitin catabolism in T. kodakaraensis. These results suggest that T. kodakaraensis, unlike other organisms, possesses a novel chitinolytic pathway where GlcNAc 2 from chitin is first deacetylated and successively hydrolyzed to glucosamine. This is the first report that reveals the primary structure of GlcNase not only from an archaeon but also from any organism.

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Section: Discussionmentioning

confidence: 99%

Characterization of an Exo-β- d -Glucosaminidase Involved in a Novel Chitinolytic Pathway from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

et al. 2003

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“…However, these uses do not often yield insight into the physiological roles of these enzymes. Although ␤-galactosidases (EC 3.2.1.23) are also found in glycoside hydrolase families (GHFs) 1, 2, and 35 (groups differing in secondary structure and other attributes [20,21]), we were especially curious about the in vivo function of GHF 42 ␤-galactosidases because they occur in organisms isolated from diverse habitats where lactose would not be present, e.g., hot springs (33,47,65), hypersaline environments (27,54), and soil (Bacillus and Streptomyces spp. ).…”

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confidence: 99%

Bioinformatic, Genetic, and Biochemical Evidence that Some Glycoside Hydrolase Family 42 β-Galactosidases Are Arabinogalactan Type I Oligomer Hydrolases

Shipkowski

Brenchley

2006

Appl Environ Microbiol

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Glycoside hydrolases are organized into glycoside hydrolase families (GHFs) and within this larger group, the ␤-galactosidases are members of four families: 1, 2, 35, and 42. Most genes encoding GHF 42 enzymes are from prokaryotes unlikely to encounter lactose, suggesting a different substrate for these enzymes. In search of this substrate, we analyzed genes neighboring GHF 42 genes in databases and detected an arrangement implying that these enzymes might hydrolyze oligosaccharides released by GHF 53 enzymes from arabinogalactan type I, a pectic plant polysaccharide. Because Bacillus subtilis has adjacent GHF 42 and GHF 53 genes, we used it to test the hypothesis that a GHF 42 enzyme (LacA) could act on the oligosaccharides released by a GHF 53 enzyme (GalA) from galactan. We cloned these genes, plus a second GHF 42 gene from B. subtilis, yesZ, into Escherichia coli and demonstrated that cells expressing LacA with GalA gained the ability to use galactan as a carbon source. We constructed B. subtilis mutants and showed that the increased ␤-galactosidase activity generated in response to the addition of galactan was eliminated by inactivating lacA or galA but unaffected by the inactivation of yesZ. As further demonstration, we overexpressed the LacA and GalA proteins in E. coli and demonstrated that these enzymes degrade galactan in vitro as assayed by thin-layer chromatography. Our work provides the first in vivo evidence for a function of some GHF 42 ␤-galactosidases. Similar functions for other ␤-galactosidases in both GHFs 2 and 42 are suggested by genomic data.␤-Galactosidases, such as the LacZ ␤-galactosidase of Escherichia coli, are used as molecular biology tools, industrial enzymes, and models for exploring structure-function relationships. However, these uses do not often yield insight into the physiological roles of these enzymes. Although ␤-galactosidases (EC 3.2.1.23) are also found in glycoside hydrolase families (GHFs) 1, 2, and 35 (groups differing in secondary structure and other attributes [20,21]), we were especially curious about the in vivo function of GHF 42 ␤-galactosidases because they occur in organisms isolated from diverse habitats where lactose would not be present, e.g., hot springs (33, 47, 65), hypersaline environments (27, 54), and soil (Bacillus and Streptomyces spp.).Over a dozen GHF 42 enzymes have been characterized, and most are ␤-galactosidases (as shown by maximal activity with the) with less than 10% relative activity on nongalactosidic chromogens (with two exceptions [27,34]). However, experimental data supporting lactose hydrolysis by GHF 42 ␤-galactosidases are absent (34, 44, 48) or weak. Several prokaryotes possessing a GHF 42 gene are unable to grow on lactose as a sole carbon source (1, 9, 27), and at least two GHF 42 ␤-galactosidases do not cleave lactose in vitro (27,64). The determination of growth on lactose can be complicated by the presence of multiple ␤-galactosidases (7,16,25) because not all of the ␤-galactosidases may be participating equally (or at all) as...

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“…During the last few years, other family 42 gene sequences have been added to the database (8,11,12,19,22,25,27). Many of these sequences have resulted from genome-sequencing projects, and there is little information about the enzymes or whether the proposed gene sequences are even transcribed in the organisms.…”

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Characterization of a Salt-Tolerant Family 42 β-Galactosidase from a Psychrophilic Antarctic Planococcus Isolate

Sheridan

Brenchley

2000

Appl Environ Microbiol

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We isolated a gram-positive, halotolerant psychrophile from a hypersaline pond located on the McMurdo Ice Shelf in Antarctica. A phylogenetic analysis of the 16S rRNA gene sequence of this organism showed that it is a member of the genus Planococcus. This assignment is consistent with the morphology and physiological characteristics of the organism. A gene encoding a ␤-galactosidase in this isolate was cloned in an Escherichia coli host. Sequence analysis of this gene placed it in glycosidase family 42 most closely related to an enzyme from Bacillus circulans. Even though an increasing number of family 42 glycosidase sequences are appearing in databases, little information about the biochemical features of these enzymes is available. Therefore, we purified and characterized this enzyme. The purified enzyme did not appear to have any metal requirement, had an optimum pH of 6.5 and an optimum temperature of activity at 42°C, and was irreversibly inactivated within 10 min when it was incubated at 55°C. The enzyme had an apparent K m of 4.9 mol of o-nitrophenyl-␤-D-galactopyranoside, and the V max was 467 mol of o-nitrophenol produced/min/mg of protein at 39°C. Of special interest was the finding that the enzyme remained active at high salt concentrations, which makes it a possible reporter enzyme for halotolerant and halophilic organisms.Glycosidases (EC 3.2.1, EC 3.2.2, and EC 3.2.3) hydrolyze the bond(s) between two or more carbohydrates or the bond between a carbohydrate moiety and a noncarbohydrate moiety. Because there are so many possible combinations of oligosaccharide isomers, there are a variety of enzymes capable of hydrolyzing these compounds. Traditionally, glycosidases were grouped together based on the ability to hydrolyze similar substrates; for example, enzymes that hydrolyze lactose or its related chromogens o-nitrophenyl-␤-D-galactopyranoside (ONPG) and 5-bromo-4-chloro-3-indoyl-␤-D-galactoside (XGal) were classified as ␤-galactosidases. One of the moststudied and useful ␤-galactosidases is the lacZ enzyme of Escherichia coli. However, many other important galactosidases are now being discovered in phylogenetically diverse organisms. The advent of computer databases containing more sequence information now makes it possible to compare and group these enzymes in order to obtain additional information about their evolutionary relationships.A useful classification for glycosidases was developed by Henrissat and coworkers (4, 6, 9, 10), who organized these enzymes into different families based on amino acid sequence similarities and hydrophobic cluster analysis data. This classification identifies possible common structural domains, thereby defining evolutionary connections and suggesting hydrolytic mechanisms for the glycosidases. The classification of Henrissat et al. divides enzymes that exhibit ␤-galactosidase activity and have the same Enzyme Commission designation (EC 3.2.1.23) into three distinct families. Sequence analyses of these three families have shown that the individual groups are extre...

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Thermostable β-Galactosidase from an Extreme Thermophile,Thermussp. A4: Enzyme Purification and Characterization, and Gene Cloning and Sequencing

Cited by 62 publications

References 20 publications

Characterization of an Exo-β- d -Glucosaminidase Involved in a Novel Chitinolytic Pathway from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

Characterization of an Exo-β- d -Glucosaminidase Involved in a Novel Chitinolytic Pathway from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

Bioinformatic, Genetic, and Biochemical Evidence that Some Glycoside Hydrolase Family 42 β-Galactosidases Are Arabinogalactan Type I Oligomer Hydrolases

Characterization of a Salt-Tolerant Family 42 β-Galactosidase from a Psychrophilic Antarctic Planococcus Isolate

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