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A Highly Active Protein Repair Enzyme from an Extreme Thermophile: the L-Isoaspartyl Methyltransferase from Thermotoga Maritima1

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A Highly Active Protein Repair Enzyme from an Extreme Thermophile: the L-Isoaspartyl Methyltransferase from Thermotoga Maritima1 ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 358, No. 2, October 15, pp. 222–231, 1998 Article No. BB980830 A Highly Active Protein Repair Enzyme from an Extreme Thermophile: The L-Isoaspartyl Methyltransferase from Thermotoga maritima1 Jeffrey K. Ichikawa2 and Steven Clarke3 Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, California 90095-1569 Received April 17, 1998, and in revised form June 29, 1998 data suggest that the Thermotoga enzyme has unique We show that the open reading frame in the Thermo- features for initiating repair in damaged proteins con- toga maritima genome tentatively identified as the taining L-isoaspartyl residues at elevated tempera- pcm gene (R. V. Swanson et al., J. Bacteriol. 178, 484– tures. © 1998 Academic Press 489, 1996) does indeed encode a protein L-isoaspartate Key Words: L-isoaspartate (D-aspartate) O-methyl- (D-aspartate) O-methyltransferase (EC 2.1.1.77) and transferase; Thermotoga maritima; thermophile; pro- that this protein repair enzyme displays several novel tein repair. features. We expressed the 317 amino acid pcm gene product of this thermophilic bacterium in Escherichia coli as a fusion protein with an N-terminal 20 residue hexa-histidine-containing sequence. This protein con- Particular enzymes that have been conserved tains a C-terminal domain of approximately 100 resi- throughout evolution have been referred to as “first dues not previously seen in this enzyme from various edition” proteins (1). These enzymes are thought to prokaryotic or eukaryotic species and which does not catalyze the essential pathways that have been con- have sequence similarity to any other entry in the served over the billions of years of evolutionary history GenBank databases. The C-terminal region appears to that separate bacteria and humans. As new gene and be required for enzymatic function as no activity is genome sequences are reported for organisms repre- detected in two recombinant constructs lacking this sentative of the deepest roots of the evolutionary tree, domain. Sedimentation equilibrium analysis indi- we begin to see what the “first printing” of these en- cated that the enzyme is monomeric in solution. The zymes might have been like (2, 3). Km values for measured for peptide and protein sub- strates were found to be intermediate between those One such enzyme, the protein L-isoaspartyl (D-aspar- of the high-affinity human enzyme and those of the tyl) O-methyltransferase (EC 2.1.1.77), has been iden- lower-affinity wheat, nematode, and E. coli enzymes. tified in a wide variety of organisms, including mam- The enzyme was extremely heat stable, with no loss of mals, nematodes, insects, plants, and bacteria (4–10) activity after 60 min at 100°C. Enzyme activity was and catalyzes the transfer of a methyl group from observed at temperatures as high as 93°C with an op- S-adenosylmethionine (AdoMet) to the a-carboxyl timal activity of 164 nmol/min/mg protein at 85°C. This group of L-isoaspartyl residues (11). These residues activity is approximately 18-fold higher than the max- result from the spontaneous deamidation of asparagi- imal activities of mesophilic homologs at 37°C. These nyl residues and isomerization of aspartyl residues (12) and can compromise the structure and function of pro- 1 This work was supported by National Institute of Health Grant teins due to the addition of a methylene group into the GM26020 and National Science Foundation Grant MCB-9305405. polypeptide backbone. The detrimental effects of isoas- Additional support was provided by National Institute of Health partyl residues on enzymatic activity have been dem- Training Grant GM07185 to J.K.I. onstrated in several proteins, including the bacterial 2 Current address: Department of Microbiology, School of Medi- cine, University of Washington, Seattle, WA 98195-7242. phosphocarrier protein, HPr (13), calmodulin (14), and 3 To whom correspondence and reprint requests should be ad- others (15–18). The methylation of these abnormal res- dressed. Fax: (310) 825-1968. E-mail: [email protected]. idues by the L-isoaspartyl methyltransferase has been 222 0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. L-ISOASPARTYL METHYLTRANSFERASE FROM Thermotoga maritima 223 shown, in vitro, to initiate the repair of these residues The purified DNA fragment was first treated with the Klenow frag- to aspartyl residues (13, 14, 19, 20). The best in vivo ment of E. coli DNA polymerase I. After heat denaturation of the Klenow fragment, the 59-end of the pcm gene was digested with evidence for the beneficial effects of repairing isoaspar- NdeI. This fragment was purified away from restriction enzymes and tyl damage has come from mouse studies, where ani- small DNA fragments using GeneClean III kit and was ligated in mals lacking this enzyme accumulate isoaspartyl dam- between the NdeI and SmaI sites of pT7-7 using T4 DNA ligase age and die of seizures at an early age (21). Further- (Gibco BRL) in a 10-ml reaction for 12 h at 4°C. The products of the more, disruption of the pcm gene in the nematode ligation reactions were transformed into E. coli strain DH5a (Gibco BRL). Insert-containing plasmid clones were initially identified by Caenorhabditis elegans has also shown to have detri- detection of a single 3.5-kbp ClaI-digested fragment on agarose gel mental effects on dauer-phase survival and reproduc- electrophoresis. The plasmids (designated pTmPCM1) containing tive fitness (22). Finally, studies in Escherichia coli the full-length pcm gene were confirmed by further restriction diges- have shown that stationary-phase cells lacking the tions. Additionally, complete nucleotide sequencing of the pcm gene and 50 bp flanking either end of it in this plasmid showed that it was L-isoaspartyl methyltransferase have a greater suscep- inserted correctly and had the expected sequence (24). tibility to oxidative and other stresses (23). The T. maritima pcm gene from pTmPCM1 was then subcloned Swanson et al. identified an open reading frame in into a plasmid that contains the E. coli pcm gene fused to an addi- the thermophilic bacterium Thermotoga maritima that tional sequence at the N-terminus encoding a hexa-histidine se- encodes a potential protein with amino acid sequence quence and a thrombin cleavage site that facilitated purification. We chose to move the T. maritima pcm gene into a new plasmid rather similarity to the protein L-isoaspartyl methyltrans- than modifying pTmPCM1 because of an additional XbaI site in the ferase from E. coli (pcm gene product, 31.2% identity) pcm coding sequence. The parent plasmid, pJI19H (Fig. 1), was and the human enzyme (25.5% identity) (24). These constructed by first replacing the XbaItoNdeI region in the vector similarities are in regions common to all protein L- portion of pJI19 with a synthetic 103-bp DNA fragment designed to isoaspartyl methyltransferases (10, 25). To character- introduce a ribosomal binding site sequence along with an initial 20 amino acid open reading frame that would be fused to any gene ize this potentially novel type of L-isoaspartyl methyl- properly ligated into the NdeI site. This sequence is identical to the transferase, we decided to overexpress this enzyme, XbaItoNdeI region upstream of the multiple cloning site of the purify it to homogeneity, and characterize its enzy- expression vector pET-15b (Novagen, Inc.). The plasmid pJI19 was matic activity. This protein methyltransferase is of digested with XbaI and NdeI and purified by agarose gel electro- interest because T. maritima thrives in a high temper- phoresis using the GeneClean III kit as described above. Finally, the ature environment, growing optimally at 80°C and able oligonucleotide fragment containing the hexa-histidine leader coding sequence was ligated into pJI19 and transformed into DH5a as to grow at temperatures as high as 90°C (26, 27), where described above. Plasmids bearing an insert were identified by re- the spontaneous rate of protein isomerization would be striction digestion and the resulting plasmid was designated pJI19H expected to be very high. Based on the energy of acti- (Fig. 1). vation measured in model peptides, an increase in tem- The E. coli pcm gene from the pJI19H plasmid was removed by perature from 23 to 90°C would increase the rate of digestion with NdeI and ClaI and purified by agarose gel purification as described above. A fragment containing the T. maritima pcm gene isoaspartyl residue formation 910-fold (28). Thus, one was isolated by digesting pTmPCM1 with NdeI and ClaI followed by of the challenges for T. maritima and other thermo- gel purification. The ligation of the T. maritima pcm gene into the philes is to avoid the accumulation of proteins with modified pT7-7 vector resulted in plasmid pTmPCM2 (Fig. 1). altered aspartyl and asparaginyl residues, and this Two C-terminal deletion mutants of T. maritima methyltrans- methyltransferase may play an important role in this ferase were constructed to examine the role of the unique C-terminal domain. One mutant, lacking the 411 base pairs that code for the process. C-terminal domain, was constructed by digesting pTmPCM2 with XhoI and HindIII and religating to generate pTmPCM3 (Fig. 1). A second mutant was also constructed by the removal of a 461 nucle- MATERIALS AND METHODS otide BamHI to BamHI portion of pTmPCM2 to generate pTmPCM4 Construction of Thermotoga maritima L-isoaspartyl methyltrans- (Fig. 1). After digestion with the appropriate enzymes, the DNA was ferase overexpression plasmids. Two oligonucleotide primers were treated with Klenow, and the DNA fragments were self-ligated to initially designed based on the putative pcm gene sequence of Swan- closed, circular plasmids and transformed into DH5a. The modified son et al. (24) to allow PCR amplification of the full-length gene from methyltransferase amino-acid sequence encoded by the pTmPCM3 T.
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