Xiao-Feng Tang

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

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°C, a down‐shift of optimum temperature by ∼15°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°C for 2 h. Notably, RTN29 exhibited a lower hydrolytic activity toward suc‐AAPF‐pNA, due to an increase in Km and a decrease in kcat, 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 enzymes 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. Biotechnol. Bioeng. 2009; 104: 862–870.

The roles of surface loop insertions and disulfide bond in the stabilization of thermophilic WF146 protease.

Thermophilic WF146 protease possesses four surface loop insertions and a disulfide bond, resembling its psychrophilic (subtilisins S41 and S39) and mesophilic (subtilisins SSII and sphericase) homologs. Deletion of the insertion 3 (positions 193–197) or insertion 4 (positions 210–221) of WF146 protease resulted in a significant decrease of the enzyme stability. In addition, substitution of the residues Pro211 and Ala212 or residue Glu221 which localized in the vicinity of a Ca2+ binding site of the enzyme by the corresponding residues in subtilisin S41 remarkably reduced the half‐life of the enzyme at 70 °C, suggesting that the three residues contributed to the thermostability of the enzyme, probably by enhancing the affinity of enzyme to Ca2+. In the presence of dithiothreitol, the WF146 protease suffered excessive autolysis, indicating that the Cys52‐Cys65 disulfide bond played a critical role in stabilizing the WF146 protease against autolysis. The autolytic cleavage sites of the WF146 protease were identified to locate between residues Asn63‐Gly64 and Cys65‐Ala66 by N‐terminal amino acid analysis of the autolytic product. It was noticed that the effect of the autolytic cleavage at Asn63‐Gly64 could be compensated by the disulfide bond Cys52‐Cys65 under non‐reducing condition, and the disulfide bond cross‐linked autolytic product remained active. The apparent stabilization effect of the disulfide bond Cys52‐Cys65 in the WF146 protease might provide a rational basis for improving the stability of subtilase against autolysis by protein engineering.

Improvement of extracellular production of a thermophilic subtilase expressed in Escherichia coli by random mutagenesis of its N-terminal propeptide

Limited secretion capacity remains a drawback of using Escherichia coli as the host for the production of recombinant proteins. In this report, random mutagenesis was performed within the N-terminal propeptide of thermostable WF146 protease, a subtilase from thermophilic Bacillus sp. WF146, generating a variant named WBMMT with improved capacity for extracellular production when expressed in E. coli. Two mutations, L(-57)Q and E(-10)D, were identified within the N-terminal propeptide. The amount of WBMMT in the culture medium was found to be about three times higher than that of wild type. Besides, the introduction of mutations L(-57)Q/E(-10)D into the N-terminal propeptide also accelerated the maturation of the enzyme. Biochemical analysis indicated that the thermostability and the catalytic activity of mature WBMMT were similar to those of wild type. Far-UV CD spectra analysis and limited proteolysis experiments suggested that the mutations L(-57)Q/E(-10)D resulted in a structural change in the N-terminal propeptide of the proform, and the N-terminal propeptide became more flexible, which might be beneficial for the proform to keep in a translocation-competent state. Our result indicates that N-terminal propeptide engineering may be a valuable approach for improving extracellular production of recombinant subtilases expressed in E. coli.

The Complete Genome Sequence of Natrinema sp. J7-2, a Haloarchaeon Capable of Growth on Synthetic Media without Amino Acid Supplements

Natrinema sp. J7-2 is an extreme haloarchaeon capable of growing on synthetic media without amino acid supplements. Here we report the complete genome sequence of Natrinema sp. J7-2 which is composed of a 3,697,626-bp chromosome and a 95,989-bp plasmid pJ7-I. This is the first complete genome sequence of a member of the genus Natrinema. We demonstrate that Natrinema sp. J7-2 can use gluconate, glycerol, or acetate as the sole carbon source and that its genome encodes complete metabolic pathways for assimilating these substrates. The biosynthetic pathways for all 20 amino acids have been reconstructed, and we discuss a possible evolutionary relationship between the haloarchaeal arginine synthetic pathway and the bacterial lysine synthetic pathway. The genome harbors the genes for assimilation of ammonium and nitrite, but not nitrate, and has a denitrification pathway to reduce nitrite to N2O. Comparative genomic analysis suggests that most sequenced haloarchaea employ the TrkAH system, rather than the Kdp system, to actively uptake potassium. The genomic analysis also reveals that one of the three CRISPR loci in the Natrinema sp. J7-2 chromosome is located in an integrative genetic element and is probably propagated via horizontal gene transfer (HGT). Finally, our phylogenetic analysis of haloarchaeal genomes provides clues about evolutionary relationships of haloarchaea.

Functional Insight into the C-Terminal Extension of Halolysin SptA from Haloarchaeon Natrinema sp. J7

Halolysin SptA from haloarchaeon Natrinema sp. J7 consists of a subtilisin-like catalytic domain and a C-terminal extension (CTE) containing two cysteine residues. In this report, we have investigated the function of the CTE using recombinant enzymes expressed in Haloferax volcanii WFD11. Deletion of the CTE greatly reduced but did not abolish protease activity, which suggests that the CTE is not essential for enzyme folding. Mutational analysis suggests that residues Cys303 and Cys338 within the CTE form a disulfide bond that make this domain resistant to autocleavage and proteolysis under hypotonic conditions. Characterization of full-length and CTE-truncation enzymes indicates the CTE not only confers extra stability to the enzyme but also assists enzyme activity on protein substrates by facilitating binding at high salinities. Interestingly, homology modeling of the CTE yields a β-jelly roll-like structure similar to those seen in Claudin-binding domain of Clostridium perfringens enterotoxin (clostridial C-CPE) and collagen binding domain (CBD), and the CTE also possesses collagen-binding activity, making it a potential candidate as an anchoring unit in drug delivery systems.

Cold-adapted maturation of thermophilic WF146 protease by mimicking the propeptide binding interactions of psychrophilic subtilisin S41

Thermophilic WF146 protease matures efficiently at 60 °C, but quite slowly at low temperatures. In this report, seven amino acid residues involved in interactions between the mature domain and the propeptide of the enzyme were substituted by corresponding residues of psychrophilic subtilisin S41 to generate mutant Mut7 (S105G/G107D/Y117E/S136N/V143G/K144E/D145S). Mut3 (S105G/G107D/Y117E) and Mut4 (S136N/V143G/K144E/D145S) were also constructed. Transferring structural features from S41 endowed Mut7 with a remarkably increased maturation rate, as well as an improved caseinolytic activity at 25 °C. Moreover, Mut3 and Mut4 each obtained one of the above endowments. Further studies suggest that low‐temperature activity and maturation rate are not necessarily linked, and uncoupling structural elements modulating the two properties may be advantageous to cold adaptation.

Molecular basis for auto- and hetero-catalytic maturation of a thermostable subtilase from thermophilic Bacillus sp. WF146

Background: The autocatalytic maturation mechanisms of subtilases are well known, but little is known about their hetero-catalytic maturation. Results: The N-terminal propeptide of the WF146 protease can be removed via both cis- and trans-processing reaction-initiated maturation pathways. Conclusion: The WF146 protease is intrinsically able to mature either autocatalytically or hetero-catalytically. Significance: Our work provides new insights into maturation mechanisms of subtilases. The proform of the WF146 protease, an extracellular subtilase produced by thermophilic Bacillus sp. WF146, matures efficiently at high temperatures. Here we report that the proform, which contains an N-terminal propeptide composed of a core domain (N*) and a linker peptide, is intrinsically able to mature via multiple pathways. One autocatalytic pathway is initiated by cis-processing of N* to generate an autoprocessed complex N*-IWT, and this step is followed by truncation of the linker peptide and degradation of N*. Another autocatalytic pathway is initiated by trans-processing of the linker peptide followed by degradation of N*. Unlike most reported subtilases, the maturation of the WF146 protease occurs not only autocatalytically but also hetero-catalytically whereby heterogeneous proteases accelerate the maturation of the WF146 protease via trans-processing of the proform and N*-IWT. Although N* acts as an intramolecular chaperone and an inhibitor of the mature enzyme, the linker peptide is susceptible to proteolysis, allowing the trans-processing reaction to occur auto- and hetero-catalytically. These studies also demonstrate that the WF146 protease undergoes subtle structural adjustments during the maturation process and that the binding of Ca2+ is required for routing the proform to mature properly at high temperatures. Interestingly, under Ca2+-free conditions, the proform is cis-processed into a unique propeptide-intermediate complex (N*-IE) capable of re-synthesis of the proform. Based on the basic catalytic principle of serine proteases and these experimental results, a mechanism for the cis-processing/re-synthesis equilibrium of the proform and the role of the linker peptide in regulation of this equilibrium has been proposed.

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

ABSTRACT The 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 half-life 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 (Tm ) 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 Km 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.

Chitin Accelerates Activation of a Novel Haloarchaeal Serine Protease That Deproteinizes Chitin-Containing Biomass

ABSTRACT The haloarchaeon Natrinema sp. strain J7-2 has the ability to degrade chitin, and its genome harbors a chitin metabolism-related gene cluster that contains a halolysin gene, sptC. The sptC gene encodes a precursor composed of a signal peptide, an N-terminal propeptide consisting of a core domain (N*) and a linker peptide, a subtilisin-like catalytic domain, a polycystic kidney disease domain (PkdD), and a chitin-binding domain (ChBD). Here we report that the autocatalytic maturation of SptC is initiated by cis-processing of N* to yield an autoprocessed complex (N*-IWT), followed by trans-processing/degradation of the linker peptide, the ChBD, and N*. The resulting mature form (MWT) containing the catalytic domain and the PkdD showed optimum azocaseinolytic activity at 3 to 3.5 M NaCl, demonstrating salt-dependent stability. Deletion analysis revealed that the PkdD did not confer extra stability on the enzyme but did contribute to enzymatic activity. The ChBD exhibited salt-dependent chitin-binding capacity and mediated the binding of N*-IWT to chitin. ChBD-mediated chitin binding enhances SptC maturation by promoting activation of the autoprocessed complex. Our results also demonstrate that SptC is capable of removing proteins from shrimp shell powder (SSP) at high salt concentrations. Interestingly, N*-IWT released soluble peptides from SSP faster than did MWT. Most likely, ChBD-mediated binding of the autoprocessed complex to chitin in SSP not only accelerates enzyme activation but also facilitates the deproteinization process by increasing the local protease concentration around the substrate. By virtue of these properties, SptC is highly attractive for use in preparation of chitin from chitin-containing biomass.

Characterization of the Haloarcula hispanica amyH gene promoter, an archaeal promoter that confers promoter activity in Escherichia coli.

Archaea form a third domain of life that is distinct from Bacteria and Eukarya. According to the current knowledge, the basal transcription machinery of Archaea (including the core promoter architecture, the RNA polymerase, and the basal transcription factors) closely resembles that of Eukarya in structure and function, while differing considerably from the bacterial paradigm. In the present study, the promoter region of the halophilic archaeon Haloarcula hispanicas amyH gene was isolated and characterized, and it was surprisingly revealed that the amyH gene promoter could confer promoter activity (i.e., drive transcription) in haloarchaea (Archaea) as well as in Escherichia coli (Bacteria), where the transcriptions driven are initiated at the same adenine base. Further investigation revealed that the core structure of the amyH gene promoter possesses a combination of the typical structural characteristics of archaeal promoter, which are eukaryotic-like, and those of bacterial promoter. Our results indicate that the core promoter structures of some archaeal genes may possess a combination of eukaryotic- and bacterial-like features, and moreover, suggest a possible evolutionary relationship between basal transcription signals and transcription mechanisms of Archaea and the other two domains of life.