Glycosyl hydrolases from hyperthermophilic microorganisms

Bauer, Michael; Driskill, Lance E.; Kelly, Robert M.

doi:10.1016/s0958-1669(98)80106-7

Cited by 49 publications

(23 citation statements)

References 40 publications

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“…MPG synthase and MPG phosphatase had maximum activity at very high temperatures but no detectable activity at low temperatures, as might be expected from results with other intracellular enzymes from hyperthermophilic organisms (35,45). More interesting is the low thermostability of both enzymes at 98°C, the optimum temperature for growth of the organism.…”

Section: Pathway For Synthesis Of Mannosylglycerate In P Horikoshii supporting

confidence: 62%

Pathway for the Synthesis of Mannosylglycerate in the Hyperthermophilic Archaeon Pyrococcus horikoshii

Empadinhas¹,

Marugg²,

Borges³

et al. 2001

Journal of Biological Chemistry

111

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The biosynthetic pathway for the synthesis of the compatible solute ␣-mannosylglycerate in the hyperthermophilic archaeon Pyrococcus horikoshii is proposed based on the activities of purified recombinant mannosyl-3-phosphoglycerate (MPG) synthase and mannosyl-3-phosphoglycerate phosphatase. The former activity was purified from cell extracts, and the N-terminal sequence was used to identify the encoding gene in the completely sequenced P. horikoshii genome. This gene, designated PH0927, and a gene immediately downstream (PH0926) were cloned and overexpressed in Escherichia coli. The recombinant product of gene PH0927 catalyzed the synthesis of ␣-mannosyl-3-phosphoglycerate (MPG) from GDP-mannose and D-3-phosphoglycerate retaining the configuration about the anomeric carbon, whereas the recombinant gene product of PH0926 catalyzed the dephosphorylation of mannosyl-3-phosphoglycerate to yield the compatible solute ␣-mannosylglycerate. The MPG synthase and the MPG phosphatase were specific for these substrates. Two genes immediately downstream from mpgs and mpgp were identified as a putative bifunctional phosphomannose isomerase/mannose-1-phosphateguanylyltransferase (PH0925) and as a putative phosphomannose mutase (PH0923). Genes PH0927, PH0926, PH0925, and PH0923 were contained in an operon-like structure, leading to the hypothesis that these genes were under the control of an unknown osmosensing mechanism that would lead to ␣-mannosylglycerate synthesis. Recombinant MPG synthase had a molecular mass of 45,208 Da, a temperature for optimal activity between 90 and 100°C, and a pH optimum between 6.4 and 7.4; the recombinant MPG phosphatase had a molecular mass of 27,958 Da and optimum activity between 95 and 100°C and between pH 5.2 and 6.4. This is the first report of the characterization of MPG synthase and MPG phosphatase and the elucidation of a pathway for the synthesis of mannosylglycerate in an archaeon.

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Section: Pathway For Synthesis Of Mannosylglycerate In P Horikoshii supporting

confidence: 62%

Pathway for the Synthesis of Mannosylglycerate in the Hyperthermophilic Archaeon Pyrococcus horikoshii

Empadinhas¹,

Marugg²,

Borges³

et al. 2001

Journal of Biological Chemistry

111

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“…Table 1 shows the distribution of these enzymes in various hyperthermophilic microorganisms; glycosyl hydrolases have been classified according to the nomenclature scheme suggested by Henrissat et al (27). Since many hyperthermophilic organisms utilize complex carbohydrates as carbon and energy sources, it is apparent that multienzyme systems are needed to hydrolyze polysaccharides that are too large to be transported across the cell membrane (3,16). As a result, many endo-acting glycosidases are cell membrane associated or completely secreted (14).…”

Section: Resultsmentioning

confidence: 99%

Regulation of Endo-Acting Glycosyl Hydrolases in the Hyperthermophilic Bacterium Thermotoga maritima Grown on Glucan- and Mannan-Based Polysaccharides

Chhabra

Shockley

Ward

et al. 2002

Appl Environ Microbiol

102

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The genome sequence of the hyperthermophilic bacterium Thermotoga maritima encodes a number of glycosyl hydrolases. Many of these enzymes have been shown in vitro to degrade specific glycosides that presumably serve as carbon and energy sources for the organism. However, because of the broad substrate specificity of many glycosyl hydrolases, it is difficult to determine the physiological substrate preferences for specific enzymes from biochemical information. In this study, T. maritima was grown on a range of polysaccharides, including barley ␤-glucan, carboxymethyl cellulose, carob galactomannan, konjac glucomannan, and potato starch. In all cases, significant growth was observed, and cell densities reached 10 9 cells/ml. Northern blot analyses revealed different substrate-dependent expression patterns for genes encoding the various endo-acting ␤-glycosidases; these patterns ranged from strong expression to no expression under the conditions tested. For example, cel74 (TM0305), a gene encoding a putative ␤-specific endoglucananse, was strongly expressed on all substrates tested, including starch, while no evidence of expression was observed on any substrate for lam16 (TM0024), xyl10A (TM0061), xyl10B (TM0070), and cel12A (TM1524), which are genes that encode a laminarinase, two xylanases, and an endoglucanase, respectively. The cel12B (TM1525) gene, which encodes an endoglucanase, was expressed only on carboxymethyl cellulose. An extracellular mannanase encoded by man5 (TM1227) was expressed on carob galactomannan and konjac glucomannan and to a lesser extent on carboxymethyl cellulose. An unexpected result was the finding that the cel5A (TM1751) and cel5B (TM1752) genes, which encode putative intracellular, ␤-specific endoglucanases, were induced only when T. maritima was grown on konjac glucomannan. To investigate the biochemical basis of this finding, the recombinant forms of Man5 (M r , 76,900) and Cel5A (M r , 37,400) were expressed in Escherichia coli and characterized. Man5, a T. maritima extracellular enzyme, had a melting temperature of 99°C and an optimun temperature of 90°C, compared to 90 and 80°C, respectively, for the intracellular enzyme Cel5A. While Man5 hydrolyzed both galactomannan and glucomannan, no activity was detected on glucans or xylans. Cel5A, however, not only hydrolyzed barley ␤-glucan, carboxymethyl cellulose, xyloglucan, and lichenin but also had activity comparable to that of Man5 on galactomannan and higher activity than Man5 on glucomannan. The biochemical characteristics of Cel5A, the fact that Cel5A was induced only when T. maritima was grown on glucomannan, and the intracellular localization of Cel5A suggest that the physiological role of this enzyme includes hydrolysis of glucomannan oligosaccharides that are transported following initial hydrolysis by extracellular glycosidases, such as Man5.Genome sequence information for hyperthermophiles has provided significant insights into the metabolic features of these microorganisms (40). With respect to the physiology of growth of...

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“…Like ␤-amylases, glucoamylases are rare in thermophiles and hyperthermophiles. Glucoamylases have been purified from only a few anaerobes, and a putative glucoamylase gene has been identified in the M. jannaschii genome (21). Extensive work remains to determine if the putative M. jannaschii glucoamylase gene is functional and if its product has catalytic properties close to the properties required for starch saccharification.…”

Section: Applications In Starch Processingmentioning

confidence: 99%

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

Vieille¹,

Zeikus

2001

Microbiol Mol Biol Rev

1,870

1,476

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Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of >80°C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described

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Glycosyl hydrolases from hyperthermophilic microorganisms

Cited by 49 publications

References 40 publications

Pathway for the Synthesis of Mannosylglycerate in the Hyperthermophilic Archaeon Pyrococcus horikoshii

Pathway for the Synthesis of Mannosylglycerate in the Hyperthermophilic Archaeon Pyrococcus horikoshii

Regulation of Endo-Acting Glycosyl Hydrolases in the Hyperthermophilic Bacterium Thermotoga maritima Grown on Glucan- and Mannan-Based Polysaccharides

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

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