Overexpression and Characterization of a Carboxypeptidase from the Hyperthermophilic Archaeon Thermococcus Sp
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Biosci. Biotechnol. Biochem., 70 (5), 1140–1147, 2006 Overexpression and Characterization of a Carboxypeptidase from the Hyperthermophilic Archaeon Thermococcus sp. NA1 Hyun Sook LEE, Yun Jae KIM, Seung Seob BAE, Jeong Ho JEON, y Jae Kyu LIM, Sung Gyun KANG, and Jung-Hyun LEE Korea Ocean Research & Development Institute, Ansan P.O. Box 29, Seoul 425-600, Korea Received September 14, 2005; Accepted January 11, 2006 Genomic analysis of a hyperthermophilic archaeon, peptidase Taq),2) T. thermophilus,3) and the hyperther- Thermococcus sp. NA1, revealed the presence of an mophilic archaeon Pyrococcus furiosus4) have been 1,497 bp open reading frame, encoding a protein of 499 purified and characterized so far. Thermostable carboxy- amino acids. The deduced amino acid sequence was peptidase 1 is known to be very thermostable, with a similar to thermostable carboxypeptidase 1 from Pyro- temperature optimum of 80–100 C.2,4) High optimum coccus furiosus, a member of peptidase family M32. Five temperatures for activity have been reported for other motifs, including the HEXXH motif with two histidines carboxypeptidases purified from the bacterium Ther- coordinated with the active site metal, were conserved. moactinomyces vulgaris5) and the archaeon Sulfolobus The carboxypeptidase gene was cloned and overex- solfataricus,6) with temperature optima of 60 and 85 C pressed in Escherichia coli. Molecular masses assessed respectively. by SDS–PAGE and gel filtration were 61 kDa and Thermostable carboxypeptidase 1 exhibits broad sub- 125 kDa respectively, which points to a dimeric struc- strate specificities. For instance, P. furiosus CP releases ture for the recombinant enzyme, designated TNA1 CP. basic, aromatic, neutral, and polar amino acids from the The enzyme showed optimum activity toward Z-Ala- C-terminus while it is unable to digest peptides with C- À1 À1 4) Arg at pH 6.5 and 70–80 C(kcat=Km ¼ 8:3 mM s ). terminal Gly, Pro, or acidic residues (Asp and Glu). In In comparison with that of P. furiosus CP (kcat=Km ¼ the case of carboxypeptidase Taq, it cleaves C-terminal 667 mMÀ1 sÀ1), TNA1 CP exhibited 80-fold lower cata- neutral, basic, and acidic amino acids most readily, with lytic efficiency. The enzyme showed broad substrate the exception of Pro and hydrolyses amino acids with specificity with a preference for basic, aliphatic, and long side chains.2) aromatic C-terminal amino acids. This broad specificity The biological role of microbial metallocarboxypep- was confirmed by C-terminal ladder sequencing of tidases has not been established, while mammalian porcine N-acetyl-renin substrate by TNA1 CP. metallocarboxypeptidases play key roles in major bio- logical processes, ranging from digestive-protein degra- Key words: genomic analysis; hyperthermophile; clon- dation, as effected by carboxypeptidase A and B,7,8) to ing; expression; carboxypeptidase specific proteolytic processing, as in the maturation of biologically active peptides, as effected by carboxypep- Carboxypeptidases (CPs) act at C-terminals of the tidase N and enkephalin convertase.9,10) It has been polypeptide chain and liberate a single amino acid. They suggested that the broad specificities of P. furiosus CP can be divided into three major groups, serine carboxy- against synthetic and natural peptide substrates might peptidases, metallocarboxypeptidases, and cysteine car- play a role in digestion and protein turnover as opposed boxypeptidases, based on the nature of the amino acid to specific post-translational modification as seen in residues at the active site of the enzymes. higher organisms.4) Metallocarboxyproteases are characterized by the There is growing interest in thermostable enzymes requirement of a divalent metal ion for their activity. because of their broad range of biotechnological Thermostable carboxypeptidase 1 (EC 3.4.17.19), be- applications and as useful models to understand better longing to peptidase family M32, is distinct from other the structural basis of thermostability.11,12) The broad metallocarboxypeptidases, since it contains a HEXXH substrate specificities of thermostable carboxypeptidase metal-binding motif.1) Several thermostable carboxy- 1 allow their use in C-terminal sequencing of pro- peptidase 1 genes that span all three kingdoms of life, teins.2,4) Using N-acetyl-renin substrate, P. furiosus CP namely archaea, bacteria, and eucarya, have been was able to sequence up to seven residues from the C- identified. Among them, the only three thermostable terminus releasing Leu, Val, Phe, Tyr, Ser, and His carboxypeptidase 1 from Thermus aquaticus (carboxy- residues. y To whom correspondence should be addressed. Tel: +82-31-400-6243; Fax: +82-31-406-2495; E-mail: [email protected] Characterization of an Archaeal Carboxypeptidase 1141 Recently, we isolated a hyperthermophilic archaeon, 1 h at 4 C). To purify TNA1 CP with the His6-Tag, Thermococcus sp. NA1, and the whole genome se- the resulting supernatant was applied to a column of quence was determined in order to search for valuable, TALONÔ metal affinity resin (BD Biosciences Clon- extremely thermostable enzymes. Analysis of the ge- tech, Palo Alto, CA) and washed with 10 mM imidazole nome information of Thermococcus sp. NA1 showed a (Sigma, St. Louis, MO) in 50 mM Tris–HCl buffer (pH carboxypeptidase gene belonging to thermostable car- 8.0) containing 0.1 M KCl and 10% glycerol, TNA1 CP boxypeptidase 1. In this study, we describe the cloning was eluted with 300 mM imidazole in the buffer. To of the gene and characterization of the recombinant purify TNA1 CP (no tag), the supernatant was applied to enzyme with regard to its enzymatic properties. a column of HiTrap Q (5 ml, Amersham Biosciences) equilibrated with 50 mM Tris–HCl (pH 7.5). A gradient Materials and Methods from 0 to 1 M KCl in the same buffer was applied to the column. The active fractions were pooled and concen- Strains and growth conditions. E. coli DH5 was trated using Centricon YM-10 (Millipore, Bedford, used for plasmid propagation and nucleotide sequenc- MA). The sample was applied to a Superdex 200 ing. E. coli BL21-CodonPlus(DE3)-RIL cells (Strata- 10/300 GL column (Amersham Biosciences) equili- gene, LaJolla, CA) and plasmid pET-24a(þ) (Novagen, brated with 50 mM Tris–HCl (pH 8.0) and 0.15 M NaCl. Madison, WI) were used for gene expression. E. coli The active fractions of TNA1 CP and TNA1 CP (no tag) strains were cultivated in Luria-Bertani medium at were then buffer exchanged to 50 mM Tris–HCl buffer 37 C, and kanamycin was added to the medium at a (pH 8.0) containing 10% glycerol using Centricon final concentration of 50 mg/ml. YM-10. The protein concentration was estimated from the DNA manipulation and sequencing. DNA manipula- absorbance at 280 nm using an extinction coefficient of À À tions were performed by standard procedures, as 90,750 M 1 cm 1. The protein purity was examined described by Sambrook and Russell.13) Restriction by sodium dodecyl sulfate–polyacrylamide gel electro- enzymes and other modifying enzymes were purchased phoresis (SDS–PAGE) analysis performed according to from Promega (Madison, WI). Small-scale preparation the standard procedure. Gel filtration of the pure enzyme of plasmid DNA from E. coli cells was performed with a was performed on a Superdex 200 10/300 GL column plasmid mini kit (Qiagen, Hilden, Germany). DNA using fast protein liquid chromatography (A¨ kta FPLC sequencing was performed with the automated sequenc- System, Amersham Biosciences), equilibrated with 50 er (ABI3100) using a BigDye terminator kit (PE Applied mM Tris–HCl (pH 8.0) and 0.2 M KCl. Thirty mg protein Biosystems, Foster City, CA). was loaded and eluted at a flow rate of 0.4 ml/min. Cloning and expression of the carboxypeptidase- Enzyme assays. TNA1 CP activity was assayed by encoding gene. Based on the whole genome sequence measuring the hydrolysis of N-Cbz-Alanyl-Arginine (Z- of Thermococcus sp. NA1, the full length of a Ala-Arg) (Bachem AG, Bubendorf, Switzerland). The carboxypeptidase gene (TNA1 CP) flanked by the NdeI assay mixture (250 ml) contained 50 mM KMES (potas- and HindIII sites was amplified by PCR with genomic sium 2-[N-morpholino]ethanesulfonic acid) buffer (pH 0 DNA and three primers (sense [5 -CGACCCGGCAT- 6.5), 0.4 mM CoCl2, and 8 mM substrate. The reaction ATGGAGGAAGTTTTCCAGAACGAAACC-30], anti- was initiated by addition of the enzyme and was sense 1 [50-CTCCACATAAGCTTGAGGTACCTCTCC- incubated at 80 C for 10 min, followed by quenching TTCACCCAGCG-30], and antisense 2 [50-CTCCACA- on ice. Subsequently, 600 ml of cadmium-ninhydrin TAAGCTTTCAGAGGTACCTCTCCTTCACCCAGCG- reagent was added, and the samples were incubated for 30]; the italicized sequences indicate the NdeI site in an additional 5 min at 80 C for color development, the sense primer and the HindIII site in the antisense followed by quenching on ice.14) The absorbance was primers). The amplified DNA fragments were digested read at 500 nm and the enzyme activity was calculated with NdeI and HindIII. The fragments were then ligated using the arginine standard curve. One unit of activity with NdeI/HindIII digested pET-24a(þ), and the result- was defined as the amount of enzyme that liberates ing recombinant plasmids were used to transform E. coli 1 mmol of arginine from Z-Ala-Arg per min at 80 C. DH5 . The recombinant plasmids were introduced Activity assays for other Z-Ala-X amino acid substrates into BL21-CodonPlus(DE3)-RIL for expression after (X = Ala, Asp, His, Leu, Met, Asn, Pro, Val, and Tyr) sequence confirmation. Overexpression was induced were performed under otherwise identical assay con- by the addition of isopropyl- -D-thiogalactopyranoside ditions. Appropriate amino acids were used to prepare (IPTG) at the mid-exponential growth phase and standard curves. incubation for 3 h at 37 C. The cells were harvested by centrifugation (6;000 Â g for 20 min at 4 C) and C-Terminal ladder sequencing of N-acetyl rennin resuspended in 50 mM Tris–HCl buffer (pH 8.0) con- substrate with TNA1 CP.