The crystal structures of the psychrophilic subtilisin S41 and the mesophilic subtilisin Sph reveal the same calcium‐loaded state

Almog, Orna; González, Ana; Godin, Noa; Leeuw, Marina de; Mekel, Marlene; Klein, D.; Braun, Sergei; Shoham, G.; Walter, R.L.

doi:10.1002/prot.22175

Cited by 27 publications

(21 citation statements)

References 49 publications

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“…As mentioned above, all the six mutations (S100P, G108S, D114G, M137T, T153A, and S246N) identified in the coldadapted variants of WF146 protease occurred within or near the substrate-binding region, which is structurally superimposable with the corresponding region of thermophilic Tk-subtilisin (Tanaka et al, 2007), mesophilic subtilisin BPN' (Wright et al, 1969) or psychrophilic S41 (Almog et al, 2009) (Fig. S1).…”

Section: S246nmentioning

confidence: 90%

Improvement of low‐temperature caseinolytic activity of a thermophilic subtilase by directed evolution and site‐directed mutagenesis

Zhong

Song

Fang

et al. 2009

Biotech & Bioengineering

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By directed evolution and subsequent site-directed mutagenesis, cold-adapted variants of WF146 protease, a thermophilic subtilase, have been successfully engineered. A four-amino acid substitution variant RTN29 displayed a sixfold increase in caseinolytic activity in the temperature range of 15-25 degrees C, a down-shift of optimum temperature by approximately 15 degrees C, as well as a decrease in thermostability, indicating it follows the general principle of trade-off between activity and stability. Nevertheless, to some extent RTN29 remained its thermophilic nature, and no loss of activity was observed after heat-treatment at 60 degrees C for 2 h. Notably, RTN29 exhibited a lower hydrolytic activity toward suc-AAPF-pNA, due to an increase in K(m) and a decrease in k(cat), in contrast to other artificially cold-adapted subtilases with increased low-temperature activity toward small synthetic substrates. All mutations (S100P, G108S, D114G, M137T, T153A, and S246N) identified in the cold-adapted variants occurred within or near the substrate-binding region. None of these mutations, however, match the corresponding sites in naturally psychrophilic and other artificially cold-adapted subtilases, implying there are multiple routes to cold adaptation. Homology modeling and structural analysis demonstrated that these mutations led to an increase in mobility of substrate-binding region and a modulation of substrate specificity, which seemed to account for the improvement of the enzyme's catalytic activity toward macromolecular substrates at lower temperatures. Our study may provide valuable information needed to develop enzymes coupling high stability and high low-temperature activity, which are highly desired for industrial use.

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Section: S246nmentioning

confidence: 90%

Improvement of low‐temperature caseinolytic activity of a thermophilic subtilase by directed evolution and site‐directed mutagenesis

Zhong

Song

Fang

et al. 2009

Biotech & Bioengineering

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“…BglU displays typical properties of cold-adapted enzymes, including high catalytic efficiency at low temperatures and relative instability at high temperatures. Its optimal temperature is 25°C, and its activity decreases greatly within 30 min at higher temperatures (18).Many studies and hypotheses since 2000 have addressed the structural basis of cold adaptation in psychrophilic proteins (19)(20)(21)(22). However, structural data for psychrophilic enzymes remain quite limited, and no such data are available for cold-adapted BGs.…”

mentioning

confidence: 99%

“…Many studies and hypotheses since 2000 have addressed the structural basis of cold adaptation in psychrophilic proteins (19)(20)(21)(22). However, structural data for psychrophilic enzymes remain quite limited, and no such data are available for cold-adapted BGs.…”

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confidence: 99%

Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus

Miao

Hou

Fan

et al. 2016

Appl Environ Microbiol

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bPsychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic ␤-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (T m ). Mutation of His299 to Tyr increased the optimal temperature, the T m , and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures.T he ␤-glucosidases (BGs; ␤-D-glucoside glycohydrolases, EC 3.2.1.21) are a widely occurring group of enzymes that cleave the carbohydrate moiety of short-chain oligosaccharides (two to six degrees of polymerization), alkyl, and aryl ␤-glucosides (1, 2). BGs have been classified into 135 glycoside hydrolase (GH) families, based on amino acid sequence similarity. GHs can also be classified into two major types, retaining and inverting enzymes, according to changes in anomeric configuration during hydrolytic reactions. Early studies of BGs were focused on their role in degrading the plant polymers cellulose and xylan (3, 4). More recently, these enzymes have gained commercial significance because of their biotechnological application in processes related to preparation of plant-based foods, e.g., conversion of phytoestrogen glucosides in fruits and vegetables to aglycone moieties, detoxification of cassava, and degradation of bitter compounds in citrus fruit juices and unripe olives (5, 6). Many mesophilic and thermophilic BGs have been cloned, purified, and characterized during the past 2 decades (5, 7-10). In contrast to mesophilic or thermophilic homologues, BGs from organisms adapted to cold...

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“…Notably, PBL5X was not only more active but also more thermostable than the WT. The stabilizing forces observed in PBL5X, such as the additional hydrogen bonds (S136N), were conserved in S41 and/or Sph, as elucidated from their crystal structures (38,39). This raises an interesting question as to why these stabilizing forces were preserved in a heat-labile enzyme, such as psychrophilic S41, but were missing in a thermostable enzyme, such as the WF146 protease.…”

Section: Discussionmentioning

confidence: 99%

“…Notably, the WF146 protease shares high amino acid sequence identities (65 to 68%) with psychrophilic subtilisins S41 (32-34) and S39 (35), as well as with mesophilic subtilisin SSII (36,37) and sphericase (Sph) (38). The crystal structures of S41 (39) and Sph (38) have been determined. The WF146 protease, Sph, and S41 constitute a trio of thermophilic, mesophilic, and psychrophilic representatives with high sequence identity, thus making them ideal for the investigation of the temperature adaptation mechanisms of enzymes.…”

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confidence: 99%

Improving the Thermostability and Activity of a Thermophilic Subtilase by Incorporating Structural Elements of Its Psychrophilic Counterpart

Bin

Dai

Chen

et al. 2015

Appl Environ Microbiol

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bThe incorporation of the structural elements of thermostable enzymes into their less stable counterparts is generally used to improve enzyme thermostability. However, the process of engineering enzymes with both high thermostability and high activity remains an important challenge. Here, we report that the thermostability and activity of a thermophilic subtilase were simultaneously improved by incorporating structural elements of a psychrophilic subtilase. There were 64 variable regions/residues (VRs) in the alignment of the thermophilic WF146 protease, mesophilic sphericase, and psychrophilic S41. The WF146 protease was subjected to systematic mutagenesis, in which each of its VRs was replaced with those from S41 and sphericase. After successive rounds of combination and screening, we constructed the variant PBL5X with eight amino acid residues from S41. The halflife of PBL5X at 85°C (57.1 min) was approximately 9-fold longer than that of the wild-type (WT) WF146 protease (6.3 min). The substitutions also led to an increase in the apparent thermal denaturation midpoint temperature (T m ) of the enzyme by 5.5°C, as determined by differential scanning calorimetry. Compared to the WT, PBL5X exhibited high caseinolytic activity (25 to 95°C) and high values of K m and k cat (25 to 80°C). Our study may provide a rational basis for developing highly stable and active enzymes, which are highly desired in industrial applications. Many microorganisms have successfully colonized extreme temperature environments ranging from Ϫ20°C to 122°C (1, 2). Enzymes from psychrophiles, mesophiles, and (hyper)thermophiles usually perform efficient catalysis at low, moderate, and high temperatures, respectively. Psychrophilic enzymes are characterized as cold active but heat labile, and these characteristics may arise from an increase in either the global or localized flexibility of enzyme structure (3, 4). Compared to their psychrophilic and mesophilic counterparts, (hyper)thermophilic enzymes generally exhibit enhanced conformational rigidity (5-7). However, some (hyper)thermophilic enzymes may combine local flexibility in their active site with high overall rigidity, thus making them more thermostable and cold active than their mesophilic counterparts (5-7).Accumulating evidence suggests that the cumulative effect of minor improvements of local interactions enhances the intrinsic stability of (hyper)thermophilic enzymes (5, 8). Identification of protein stabilization mechanisms is normally based on comparative studies of homologous enzymes that are adapted to different temperatures, on mutational analyses, on directed evolution and on computational methods (6, 9). The results of these studies have provided a rational basis for improving enzyme stability by sitedirected mutagenesis (SDM) (10). Nevertheless, the process of engineering enzymes for higher thermostability and activity remains important and difficult. One reason for this problem is that structural differences between homologous enzymes that are adapted to different tempe...

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The crystal structures of the psychrophilic subtilisin S41 and the mesophilic subtilisin Sph reveal the same calcium‐loaded state

Cited by 27 publications

References 49 publications

Improvement of low‐temperature caseinolytic activity of a thermophilic subtilase by directed evolution and site‐directed mutagenesis

Improvement of low‐temperature caseinolytic activity of a thermophilic subtilase by directed evolution and site‐directed mutagenesis

Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus

Improving the Thermostability and Activity of a Thermophilic Subtilase by Incorporating Structural Elements of Its Psychrophilic Counterpart

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