Cleavage after residue Ala352 in the C-terminal extension is an early step in the maturation of the D1 subunit of Photosystem II in Synechocystis PCC 6803

We have investigated the pathway by which the 16 amino-acid C-terminal extension of the D1 subunit of photosystem two is removed in the cyanobacterium Synechocystis sp. PCC 6803 to leave Ala344 as the C-terminal residue. Previous work has suggested a two-step process involving formation of a processing intermediate of D1, termed iD1, of uncertain origin. Here we show by mass spectrometry that a synthetic peptide mimicking the C- terminus of the D1 precursor is cleaved by cellular extracts or purified CtpA processing protease after residue Ala352, making this a likely site for formation of iD1. Characteristics of D1 site-directed mutants with either the Leu353 residue replaced by Pro or with a truncation after Ala352 are in agreement with this assignment. Interestingly, analysis of various CtpA and CtpB null mutants further indicate that the CtpA protease plays a crucial role in forming iD1 but that, surprisingly, low levels of C-terminal processing occur in vivo in the absence of CtpA and CtpB, possibly catalysed by other related proteases. A possible role for two-step maturation of D1 in the assembly of PSII is discussed.     

Cleavage after residue Ala352 in the C-terminal extension is an early step in the maturation of the D1 subunit of Photosystem II in Synechocystis PCC 6803

We have investigated the pathway by which the 16 amino-acid C-terminal extension of the D1 subunit of photosystem two is removed in the cyanobacterium Synechocystis sp. PCC 6803 to leave Ala344 as the C-terminal residue. Previous work has suggested a two-step process involving formation of a processing intermediate of D1, termed iD1, of uncertain origin. Here we show by mass spectrometry that a synthetic peptide mimicking the C- terminus of the D1 precursor is cleaved by cellular extracts or purified CtpA processing protease after residue Ala352, making this a likely site for formation of iD1. Characteristics of D1 site-directed mutants with either the Leu353 residue replaced by Pro or with a truncation after Ala352 are in agreement with this assignment. Interestingly, analysis of various CtpA and CtpB null mutants further indicate that the CtpA protease plays a crucial role in forming iD1 but that, surprisingly, low levels of C-terminal processing occur in vivo in the absence of CtpA and CtpB, possibly catalysed by other related proteases. A possible role for two-step maturation of D1 in the assembly of PSII is discussed.     

D1蛋白酶的克隆表达、纯化、生物活性测定及多克隆抗体的制备

为了开发以D1蛋白酶作为靶标的新型除草剂,需要对先导化合物的生物活性进行检测和筛选。从菠菜中提取总RNA,逆转录合成cDNA,采用PCR扩增了CtpA的基因,连接至表达载体pET-28a中,构建了重组表达质粒,并在大肠杆菌BL21(DE3)中进行高效表达,通过降低IPTG和诱导温度获得了可溶性表达的重组蛋白酶。采用Ni-NTA亲和层析和凝胶过滤柱层析对重组蛋白进行了纯化,SDS-PAGE和Western blotting结果证实目的蛋白为含有His-tag的融合蛋白。以合成的D1前体蛋白羧基端24肽作为模拟底物,采用高效液相色谱法测定了其水解活性,结果表明活性可达1.10nmol/(mg·min),为文献报道数据的15倍。纯化后的CtpA蛋白免疫日本的长耳大白兔制备多克隆抗体,ELISA法测定其血清抗体的效价高达1:100000。该结果为抑制剂先导化合物的筛选和酶蛋白与化合物的作用机理研究提供了必要的基础。     

Pleiotropic effects of inactivating a carboxyl-terminal protease, CtpA, in Borrelia burgdorferi

A gene encoding a putative carboxyl-terminal protease (CtpA), an unusual type of protease, is present in the Borrelia burgdorferi B31 genome. The B. burgdorferi CtpA amino acid sequence exhibits similarities to the sequences of the CtpA enzymes of the cyanobacterium Synechocystis sp. strain PCC 6803 and higher plants and also exhibits similarities to the sequences of putative CtpA proteins in other bacterial species. Here, we studied the effect of ctpA gene inactivation on the B. burgdorferi protein expression profile. Total B. burgdorferi proteins were separated by two-dimensional gel electrophoresis, and the results revealed that six proteins of the wild type were not detected in the ctpA mutant and that nine proteins observed in the ctpA mutant were undetectable in the wild type. Immunoblot analysis showed that the integral outer membrane protein P13 was larger and had a more acidic pI in the ctpA mutant, which is consistent with the theoretical change in pI for P13 not processed at the carboxyl terminus. Matrix-assisted laser desorption ionization-time of flight data indicated that in addition to P13, the BB0323 protein may serve as a substrate for carboxyl-terminal processing by CtpA. Complementation analysis of the ctpA mutant provided strong evidence that the observed effect on proteins depended on inactivation of the ctpA gene alone. We show that CtpA in B. burgdorferi is involved in the processing of proteins such as P13 and BB0323 and that inactivation of ctpA has a pleiotropic effect on borrelial protein synthesis. To our knowledge, this is the first analysis of both a CtpA protease and different substrate proteins in a pathogenic bacterium.     

Different mechanisms for thermal inactivation of Bacillus subtilis signal peptidase mutants.

The type I signal peptidase SipS of Bacillus subtilis is of major importance for the processing of secretory precursor proteins. In the present studies, we have investigated possible mechanisms of thermal inactivation of five temperature-sensitive SipS mutants. The results demonstrate that two of these mutants, L74A and Y81A, are structurally stable but strongly impaired in catalytic activity at 48 degrees C, showing the (unprecedented) involvement of the conserved leucine 74 and tyrosine 81 residues in the catalytic reaction of type I signal peptidases. This conclusion is supported by the crystal structure of the homologous signal peptidase of Escherichia coli (Paetzel, M., Dalbey, R. E., and Strynadka, N. C. J. (1998) Nature 396, 186-190). In contrast, the SipS mutant proteins R84A, R84H, and D146A were inactivated by proteolytic degradation, indicating that the conserved arginine 84 and aspartic acid 146 residues are required to obtain a protease-resistant conformation. The cell wall-bound protease WprA was shown to be involved in the degradation of SipS D146A, which is in accord with the fact that SipS has a large extracytoplasmic domain. As WprA was not involved in the degradation of the SipS mutant proteins R84A and R84H, we conclude that multiple proteases are responsible for the thermal inactivation of temperature-sensitive SipS mutants.     

Mutagenesis of the NS3 Protease of Dengue Virus Type 2

The flavivirus protease is composed of two viral proteins, NS2B and NS3. The amino-terminal portion of NS3 contains sequence and structural motifs characteristic of bacterial and cellular trypsin-like proteases. We have undertaken a mutational analysis of the region of NS3 which contains the catalytic serine, five putative substrate binding residues, and several residues that are highly conserved among flavivirus proteases and among all serine proteases. In all, 46 single-amino-acid substitutions were created in a cloned NS2B-NS3 cDNA fragment of dengue virus type 2, and the effect of each mutation on the extent of self-cleavage of the NS2B-NS3 precursor at the NS2B-NS3 junction was assayed in vivo. Twelve mutations almost completely or completely inhibited protease activity, 9 significantly reduced it, 14 decreased cleavage, and 11 yielded wild-type levels of activity. Substitution of alanine at ultraconserved residues abolished NS3 protease activity. Cleavage was also inhibited by substituting some residues that are conserved among flavivirus NS3 proteins. Two (Y150 and G153) of the five putative substrate binding residues could not be replaced by alanine, and only Y150 and N152 could be replaced by a conservative change. The two other putative substrate binding residues, D129 and F130, were more freely substitutable. By analogy with the trypsin model, it was proposed that D129 is located at the bottom of the substrate binding pocket so as to directly interact with the basic amino acid at the substrate cleavage site. Interestingly, we found that significant cleavage activity was displayed by mutants in which D129 was replaced by E, S, or A and that low but detectable protease activity was exhibited by mutants in which D129 was replaced by K, R, or L. Contrary to the proposed model, these results indicate that D129 is not a major determinant of substrate binding and that its interaction with the substrate, if it occurs at all, is not essential. This mutagenesis study provided us with an array of mutations that alter the cleavage efficiency of the dengue virus protease. Mutations that decrease protease activity without abolishing it are candidates for introduction into the dengue virus infectious full-length cDNA clone with the aim of creating potentially attenuated virus stocks.     

Overexpression and characterization of carboxyl-terminal processing protease for precursor D1 protein: regulation of enzyme-substrate interaction by molecular environments

CtpA, which is classified as a novel type of serine protease with a Ser/Lys catalytic dyad, is responsible for the C-terminal processing of precursor D1 protein (pD1) of the photosystem II reaction center, a process that is indispensable for the integration of water-splitting machinery in photosynthesis. In this study, overexpression in Escherichia coli and one-step purification of spinach CtpA were carried out to analyze the characteristics of this new type of protease and to elucidate the molecular interactions in the C-terminal processing of pD1 on the thylakoid membrane. The successful accumulation of functional CtpA in E. coli may argue against the possibility, based on homology to E. coli Tsp, that the enzyme is involved in the degradation of incomplete proteins in chloroplasts, e.g. by utilizing the ssrA-tagging system. Analysis using a synthetic pD1 oligopeptide demonstrated that the enzymatic properties (including substrate recognition) of overexpressed CtpA with an extra sequence of GSHMLE at the N terminus were indistinguishable from those of the native enzyme. CtpA was insensitive to penem, which has been shown to inhibit some Ser/Lys-type proteases, suggesting that the catalytic center of CtpA is quite unique. By using the substrate in different molecular environments (i.e. synthetic pD1 oligopeptide in solution and pD1 in photosystem II-enriched thylakoid membrane), we observed a dramatic difference in the pH profile and affinity for the substrate, suggesting the presence of a specific interaction of CtpA with a factor(s) that modulates the pH dependence of proteolytic action in response to physiological conditions.     

The carboxyl-terminal extension of the precursor D1 protein of photosystem II is required for optimal photosynthetic performance of the cyanobacterium Synechocystis sp. PCC 6803

The D1 protein is an integral component of the photosystem II reaction center complex. In the cyanobacterium Synechocystis sp. PCC 6803, D1 is synthesized with a short 16-amino acids-long carboxyl-terminal extension. Removal of this extension is necessary to form active oxygen-evolving photosystem II centers. Our earlier studies have shown that this extension is cleaved by CtpA, a specific carboxyl-terminal processing protease. The amino acid sequence of the carboxyl-terminal extension is conserved among D1 proteins from different organisms, although at a level lower than that of the mature protein. In the present study we have analyzed a mutant strain of Synechocystis sp. PCC 6803 with a duplicated extension, and a second mutant that lacks the extension, to investigate the effects of these alterations on the function of the D1 protein in vivo. No significant difference in the growth rates, photosynthetic pigment composition, fluorescence induction, and oxygen evolution rates was observed between the mutants and the control strain. However, using long-term mixed culture growth analysis, we detected significant decreases in the fitness of these mutant strains. The presented data demonstrate that the carboxyl-terminal extension of the precursor D1 protein is required for optimal photosynthetic performance.     

Identification of catalytic residues of pepstatin-insensitive carboxyl proteinases from prokaryotes by site-directed mutagenesis.

Pepstatin-insensitive carboxyl proteinases from Pseudomonas sp. (PCP) and Xanthomonas sp. (XCP) have no conserved catalytic residue sequences, -Asp*-Thr-Gly- (Asp is the catalytic residue) for aspartic proteinases. To identify the catalytic residues of PCP and XCP, we selected presumed catalytic residues based on their high sequence similarity, assuming that such significant sites as catalytic residues will be generally conserved. Several Ala mutants of Asp or Glu residues were constructed and analyzed. The D170A, E222A, and D328A mutants for PCP and XD79A, XD169A, and XD348A mutants for XCP were not converted to mature protein after activation, and no catalytic activity could be detected in these mutants. The specificity constants toward chromogenic substrate of the other PCP and XCP mutants, except for the D84A mutant of PCP, were similar to that of wild-type PCP or XCP. Coupled with the result of chemical modification (Ito, M., Narutaki, S., Uchida, K., and Oda, K. (1999) J. Biochem. (Tokyo) 125, 210-216), a pair of Asp residues (170 and 328) for PCP and a pair of Asp residues (169 and 348) for XCP were elucidated to be their catalytic residues, respectively. The Glu(222) residue in PCP or Asp(79) residue in XCP was excluded from the candidates as catalytic residues, since the corresponding mutant retained its original activity.     

Oligomeric interaction of hepatitis C virus NS5B is critical for catalytic activity of RNA-dependent RNA polymerase.

HCV NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme for HCV replication, which has the "palm and fingers" substructure. We recently identified five novel residues critical for RdRP activity (Qin, W., Yamashita, T., Shirota, Y., Lin, Y., Wei, W., and Murakami, S. (2001) Hepatology 33, 728-737). Among them, GLU-18 and His-502, far from the catalytic center, may be involved in conformational change(s) for RdRP activity as addressed in some palm and fingers enzymes. We examined the possibility that NS5B is oligomerized, and we could detect the interaction between two different tagged NS5B proteins in vitro and transiently expressed in mammalian cells. By scanning 27 clustered and then point alanine substitutions in vivo and in vitro, Glu-18 and His-502 were found to be critical for the homomeric interaction in vivo and in vitro, strongly suggesting a close relationship between the oligomerization and RdRP activity of NS5B. All mutants with substitutions at these two residues failed to bind wild type NS5B, however E18H interacted with H502E in vitro and in vivo. Interestingly, the NS5B protein with E18H or H502E did not exhibit RdRP activity, but a mixture of the two mutant proteins did. These results clearly indicate that two residues of HCV NS5B are critical for the oligomerization that is prerequisite to RdRP activity.     

Specificity and role of the Borrelia burgdorferi CtpA protease in outer membrane protein processing

To further characterize the function of the Borrelia burgdorferi C-terminal protease CtpA, we used site-directed mutagenesis to alter the putative CtpA cleavage site of one of its known substrates, the outer membrane (OM) porin P13. These mutations resulted in only partial blockage of P13 processing. Ectopic expression of a C-terminally truncated P13 in B. burgdorferi indicated that the C-terminal peptide functions as a safeguard against misfolding or mislocalization prior to its proteolytic removal by CtpA. In a parallel study of Borrelia burgdorferi lipoprotein sorting mechanisms, we observed a lower-molecular-weight variant of surface lipoprotein OspC that was particularly prominent with OspC mutants that mislocalized to the periplasm or contained C-terminal epitope tags. Further investigation revealed that the variant resulted from C-terminal proteolysis by CtpA. Together, these findings indicate that CtpA rather promiscuously targets polypeptides that lack structurally constrained C termini, as proteolysis appears to occur independently of a specific peptide recognition sequence. Low-level processing of surface lipoproteins such as OspC suggests the presence of a CtpA-dependent quality control mechanism that may sense proper translocation of integral outer membrane proteins and surface lipoproteins by detecting the release of C-terminal peptides.     

Identification of active-site amino acid residues in the Chiba virus 3C-like protease

The 3C-like protease of the Chiba virus, a Norwalk-like virus, is one of the chymotrypsin-like proteases. To identify active-site amino acid residues in this protease, 37 charged amino acid residues and a putative nucleophile, Cys139, within the GDCG sequence were individually replaced with Ala in the 3BC precursor, followed by expression in Escherichia coli, where the active 3C-like protease would cleave 3BC into 3B (VPg) and 3C (protease). Among 38 Ala mutants, 7 mutants (R8A, H30A, K88A, R89A, D138A, C139A, and H157A) completely or nearly completely lost the proteolytic activity. Cys139 was replaceable only with Ser, suggesting that an SH or OH group in the less bulky side chain was required for the side chain of the residue at position 139. His30, Arg89, and Asp138 could not be replaced with any other amino acids. Although Arg8 was also not replaceable for the 3B/3C cleavage and the 3C/3D cleavage, the N-terminal truncated mutant devoid of Arg8 significantly cleaved 3CD into 3C and 3D (polymerase), indicating that Arg8 itself was not directly involved in the proteolytic cleavage. As for position 88, a positively charged residue was required because the Arg mutant showed significant activity. As deduced by the X-ray structure of the hepatitis A virus 3C protease, Arg8, Lys88, and Arg89 are far away from the active site, and the side chain of Asp138 is directed away from the active site. Therefore, these are not catalytic residues. On the other hand, all of the mutants of His157 in the S1 specificity pocket tended to retain very slight activity, suggesting a decreased level of substrate recognition. These results, together with a sequence alignment with the picornavirus 3C proteases, indicate that His30 and Cys139 are active-site residues, forming a catalytic dyad without a carboxylate directly participating in the proteolysis.     

Communication between the regulatory and the catalytic region of the cAMP-responsive guanine nucleotide exchange factor Epac

Epac1 is a guanine nucleotide exchange factor (GEF) for the small GTPase Rap1 that is directly activated by cAMP. This protein consists of a regulatory region with a cAMP-binding domain and a catalytic region that mediates the GEF activity. Epac is inhibited by an intramolecular interaction between the cAMP-binding domain and the catalytic region in the absence of cAMP. cAMP binding is proposed to induce a conformational change, which allows a LID, an alpha-helix at the C-terminal end of the cAMP-binding site, to cover the cAMP-binding site (Rehmann, H., Prakash, B., Wolf, E., Rueppel, A., de Rooij, J., Bos, J. L., and Wittinghofer, A. (2003) Nat. Struct. Biol. 10, 26-32). Here we show that mutations of conserved residues in the LID region affect cAMP binding only marginally but have a drastic effect on cAMP-induced GEF activity. Surprisingly, some of the mutants have an increased maximal GEF activity compared with wild type. Furthermore, mutation of the conserved VLVLE sequence at the C-terminal end of the LID into five alanine residues makes Epac constitutively active. From these results we conclude that the LID region plays a pivotal role in the communication between the regulatory and catalytic part of Epac.     

Isolation and characterization of monoclonal antibodies that inhibit hepatitis C virus NS3 protease

A series of mouse monoclonal antibodies (MAbs) to the nonstructural protein 3 (NS3) of hepatitis C virus was prepared. One of these MAbs, designated 8D4, was found to inhibit NS3 protease activity. This inhibition was competitive with respect to the substrate peptide (K(i) = 39 nM) but was significantly decreased by the addition of the NS4A peptide, a coactivator of the NS3 protease. 8D4 also showed marked inhibition of the NS3-dependent cis processing of the NS3/4A polyprotein but had virtually no effect on the succeeding NS3/4A-dependent trans processing of the NS5A/5B polyprotein in vitro. Epitope mapping of 8D4 with a random peptide library revealed a consensus sequence, DxDLV, that matched residues 79 to 83 (DQDLV) of NS3, a region containing the catalytic residue Asp-81. Furthermore, synthetic peptides including this sequence were shown to block the ability of 8D4 to bind to NS3, indicating that 8D4 interacts with the catalytic region of NS3. The data showing decreased inhibition potency of 8D4 against the NS3/4A complex suggest that 8D4 recognizes the conformational state of the protease active site caused by the association of NS4A with the protease.     

The critical role of the 185-189-loop in the factor Xa interaction with Na+ and factor Va in the prothrombinase complex

The S1 site (Asp(189)) of factor Xa (fXa) is located on a loop (residues 185-189) that contains three solvent-exposed charged residues (Asp(185), Lys(186), and Glu(188)) below the active-site pocket of the protease. To investigate the role of these residues in the catalytic function of fXa, we expressed three mutants of the protease in which the charges of these residues were neutralized by their substitutions with Ala (D185A, K186A, and E188A). Kinetic studies revealed that E188A has a normal catalytic activity toward small synthetic and natural substrates and inhibitors of fXa; however, the same activities were slightly ( approximately 2-fold) and dramatically ( approximately 20-50-fold) impaired for the D185A and K186A mutants, respectively. Further studies revealed that the affinity of D185A and K186A for interaction with Na(+) has also been altered, with a modest impairment ( approximately 2-fold) for the former and a dramatic impairment for the latter mutant. Both prothrombinase and direct binding studies indicated that K186A also has an approximately 6-fold impaired affinity for factor Va. Interestingly, a saturating concentration of factor Va restored the catalytic defect of K186A in reactions with prothrombin and the recombinant tick anticoagulant peptide that is known to interact with the Na(+) loop of fXa, but not with other substrates. These results suggest that factor Va interacts with 185-189-loop for fXa, which is energetically linked to the Na(+)-binding site of the protease.     

The catalytic triad of testes-specific protease 50 (TSP50) is essential for its function in cell proliferation

Testes-specific protease 50 (TSP50) is a novelly identified pro-oncogene and it shares a similar enzymatic structure with many serine proteases. Our previous results suggested that TSP50 could promote tumorigenesis through degradation of IκBα protein and activating NF-κB signaling, and the threonine mutation in its catalytic triad could depress TSP50-mediated cell proliferation. However, whether the two other residues in the catalytic triad of TSP50 play a role in maintaining protease activity and tumorigenesis, and the mechanisms involved in this process remain unclear. Here, we constructed and characterized three catalytic triad mutants of TSP50 and found that all the mutants could significantly depress TSP50-induced cell proliferation and colony formation in vitro and tumor formation in vivo, and the aspartic acid at position 206 in the catalytic triad played a more crucial role than threonine and histidine in this process. Mechanistic studies revealed that the mutants in the catalytic triad abolished the enzyme activity of TSP50, but did not change the cellular localization. Furthermore, our data indicated that all the three mutants suppressed activation of NF-κB signal by preventing the interaction between TSP50 and the NF-κB:IκBα complex. Most importantly, we demonstrated that TSP50 could interact with IκBα protein and cleave it directly as a new protease in vitro.     

Identification of domain structures in the propeptide of corin essential for the processing of proatrial natriuretic peptide

Corin is a type II transmembrane serine protease and functions as the proatrial natriuretic peptide (pro-ANP) convertase in the heart. In the extracellular region of corin, there are two frizzled-like cysteine-rich domains, eight low density lipoprotein receptor (LDLR) repeats, a macrophage scavenger receptor-like domain, and a trypsin-like protease domain at the C terminus. To examine the functional importance of the domain structures in the propeptide of corin for pro-ANP processing, we constructed a soluble corin, EKshortCorin, that consists of only the protease domain and contains an enterokinase (EK) recognition sequence at the conserved activation cleavage site. After being activated by EK, EKshortCorin exhibited catalytic activity toward chromogenic substrates but failed to cleave pro-ANP, indicating that certain domain structures in the propeptide are required for pro-ANP processing. We then constructed a series of corin deletion mutants and studied their functions in pro-ANP processing. Compared with that of the full-length corin, a corin mutant lacking frizzled 1 domain exhibited approximately 40% activity, whereas corin mutants lacking single LDLR repeat 1, 2, 3, or 4 had approximately 49, approximately 12, approximately 53, and approximately 77% activity, respectively. We also made corin mutants with a single mutation at a conserved Asp residue that coordinates Ca(2+)-binding in LDLR repeats 1, 2, 3, or 4 (D300Y, D336Y, D373Y, and D410Y) and showed that these mutants had approximately 25, approximately 11, approximately 16, and approximately 82% pro-ANP processing activity, respectively. Our results indicate that frizzled 1 domain and LDLR repeats 1-4 are important structural elements for corin to recognize its physiological substrate, pro-ANP.     

An essential tyrosine phosphatase homolog regulates cell separation, outer membrane integrity, and morphology in Caulobacter crescentus

Although reversible phosphorylation on tyrosine residues regulates the activity of many eukaryotic proteins, there are few examples of this type of regulation in bacteria. We have identified the first essential tyrosine phosphatase homolog in a bacterium, Caulobacter crescentus CtpA. ctpA mutants with altered active-site residues are nonviable, and depletion of CtpA yields chains of cells with blebbed outer membranes, linked by unresolved peptidoglycan. CtpA overexpression reduces cell curvature in a manner similar to deleting the intermediate filament protein crescentin, but it does not disrupt crescentin localization or membrane attachment. Although it has no obvious signal sequence or transmembrane-spanning domains, CtpA associates with the Caulobacter inner membrane. Immunolocalization experiments suggest that CtpA accumulates at the division site during the last quarter of the cell cycle. We propose that CtpA dephosphorylates one or more proteins involved in peptidoglycan biosynthesis or remodeling, which in turn affect cell separation, cell envelope integrity, and vibrioid morphology.     

In vivo and in vitro characterization of TEV protease mutants

Tobacco etch virus protease (TEVp) is frequently applied in the cleavage of fusion protein. However, production of TEV protease in Escherichia coli is hampered by low yield and poor solubility, and auto-cleavage of wild type TEVp gives rise to the loss-of-function. Previously it was reported that TEVp S219V displayed more stability, and TEVp variant containing T17S/N68D/I77V and double mutant L56V/S135G resulted in the enhanced production and solubility, respectively. Here, we introduced T17S/N68D/I77V in TEVp S219V to generate TEVpM1 and combined five amino acid mutations (T17S/L56V/N68D/I77V/S135G) in TEVp S219V to create TEVpM2. Among TEVp S219V, and two constructed variants, TEVpM2 displayed highest solubility and catalytic activity in vivo, using EmGFP as the solubility reporter, and the designed fusion protein as in vivo substrate containing an N-terminal hexahistidine tagged GST, a peptide sequence for thrombin and TEV cut and E. coli diaminopropionate ammonia-lyase. The purified TEVp mutants fused with double hexahistidine-tag at N and C terminus showed highest yield, solubility and cleavage efficiency. Mutations of five amino acid residues in TEVpM2 slightly altered protein secondary structure conformed by circular dichroism assay.