Molecular cloning, nucleotide sequence, and expression of a carboxypeptidase-encoding gene from the archaebacterium Sulfolobus solfataricus

Abstract

Mammalian metallocarboxypeptidases play key roles in major biological processes, such as digestive-protein degradation and specific proteolytic processing. A Sulfolobus solfataricus gene (cpsA) encoding a recently described zinc carboxypeptidase with an unusually broad substrate specificity was cloned, sequenced, and expressed in Escherichia coli. Despite the lack of overall sequence homology with known carboxypeptidases, seven homology blocks, including the Zn-coordinating and catalytic residues, were identified by multiple alignment with carboxypeptidases A, B, and T. S. solfataricus carboxypeptidase expressed in E. coli was found to be enzymatically active, and both its substrate specificity and thermostability were comparable to those of the purified S. solfataricus enzyme.Mammalian metallocarboxypeptidases play key roles in major biological processes, ranging from digestive-protein degradation, as effected by carboxypeptidases A and B (9, 13), to specific proteolytic processing, as in the maturation of biologically active peptides. This latter is the role, for instance, of carboxypeptidase N and enkephalin convertase (10,17).Little is known, however, regarding microbial carboxypeptidases. Recent studies led to the cloning and crystallization of a metallocarboxypeptidase from Thermoactinomyces vulgaris (27,29), which was shown to possess a dual substrate specificity, namely, the capacity to cleave both hydrophobic and basic amino acid residues, combining the activities of carboxypeptidases A and B, respectively. Sequence comparison revealed a sequence similarity of approximately 30% and a three-dimensional structure very similar to those of both of these mammalian enzymes (27,29). Another microbial carboxypeptidase with broad substrate specificity was also isolated recently from the thermophilic eubacterium Thermus aquaticus (16). However, no sequence data are so far available for this enzyme.The lack of any knowledge regarding archaebacterial carboxypeptidases led us to purify and characterize one such enzyme from Sulfolobus solfataricus (3), an extreme thermoacidophilic archaebacterium, isolated from volcanic hot springs, that grows optimally at 87ЊC (7). We found that this protein is a zinc metalloprotease endowed with a unique substrate specificity in that it could release basic, acidic, aromatic, and, to a lesser extent, aliphatic amino acids from artificial substrates. Also, it could withstand temperatures of up to 85ЊC and some organic solvents at up to 40ЊC. Furthermore, we found that salt bridges and the zinc ion play major roles in the kinetic thermal stabilization of this molecule (30). Here, we report the molecular cloning and complete nucleotide sequence of the carboxypeptidase-encoding gene. We show that despite the lack of overall sequence homology with known carboxypeptidases, seven blocks of homology with known metallocarboxypeptidases, spanning the Zn-coordinating histidines, can be identified. We also show that S. solfataricus carboxypeptidase expressed in Escherichia coli is enzymatically...

Characterization of an Aminoacylase from the Hyperthermophilic Archaeon Pyrococcus furiosus

“…The P. furiosus enzyme has 34,15,15,80,81,26,38,32,41, and 14% identity, respectively, with these enzymes but shows no sequence similarity with that of the AARE found in P. horikoshii (26). It does show high similarity, however, to the sequences of Sulfolobus solfataricus carboxypeptidase (16), Thermotoga maritima hippurate hydrolase (39), Campylobacter jejuni hippuricase (24), Arabidopsis thaliana (ILR1) indole-3-acetic acid amino acid hydrolase (4), and Arabidopsis thaliana JR3 protein (identities of 38,28,35,38, and 40%, respectively). The sequences of several of these enzymes are aligned with that of the P. furiosus enzyme in Fig.…”

Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

“…The amino acid sequence predicted from the gene had approximately 45% identity to CP from S. solfataricus ( Fig. 1) (13); however, most of the proposed active-site residues of CP (11,13) are not found in this amino acid sequence. In addition, the sequence had about 45% identity to aminoacylase from B. stearothermophilus (33) (Fig.…”

“…A thermostable CP is useful for high-temperature analysis of the C-terminal amino acid sequences of proteins. Recently, several thermostable CPs from the thermophilic bacteria Thermoactinomyces vulgaris (35,36) and Thermus aquaticus (27,28,29) and the thermophilic archaea Sulfolobus solfataricus (12,13,38) and Pyrococcus furiosus (8) have been characterized. Using genome sequencing for P. horikoshii (20, 21), we found two kinds of genes encoding CP-homologous proteins.…”

Giardia Duodenalis 14-3-3 Protein Is Polyglycylated by a Tubulin Tyrosine Ligase-like Member and Deglycylated by Two Metallocarboxypeptidases

“…The glutamate carboxypeptidase G2 of Pseudomonas sp. (subfamily M20A) hydrolyzes the C-terminal glutamate moiety from folic acid and its analogues (38), the tripeptidase T (M20B) of Lactococcus lacti hydrolyzes only tripeptides (39), the bacterial aminoacylhistidine dipeptidase performs hydrolysis of dipeptides (40), and the thermostable carboxypeptidase Ss1 (M20D) of the archean Sulfolobus solfataricus is able to cleave N-blocked tripeptides (41).…”