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Eukaryotic methionyl aminopeptidases: two classes of cobalt-dependent enzymes.

ABSTRACT: Using partial amino acid sequence data derived from porcine methionyl aminopeptidase (MetAP; methionine aminopeptidase, peptidase M; EC 3.4.11.18), a full-length clone of the homologous human enzyme has been obtained. The cDNA sequence contains 2569 nt with a single open reading frame corresponding to a protein of 478 amino acids. The C-terminal portion representing the catalytic domain shows limited identity with MetAP sequences from various prokaryotes and yeast, while the N terminus is rich in charged amino acids, including extended strings of basic and acidic residues. These highly polar stretches likely result in the spuriously high observed molecular mass (67 kDa). This cDNA sequence is highly similar to a rat protein, termed p67, which was identified as an inhibitor of phosphorylation of initiation factor eIF2 alpha and was previously predicted to be a metallopeptidase based on limited sequence homology. Model building established that human MetAP (p67) could be readily accommodated into the Escherichia coli MetAP structure and that the Co2+ ligands were fully preserved. However, human MetAP was found to be much more similar to a yeast open reading frame that differed markedly from the previously reported yeast MetAP. A similar partial sequence from Methanothermus fervidus suggests that this p67-like sequence is also found in prokaryotes. These findings suggest that there are two cobalt-dependent MetAP families, presently composed of the prokaryote and yeast sequences (and represented by the E. coli structure) (type I), on the one hand, and by human MetAP, the yeast open reading frame, and the partial prokaryotic sequence (type II), on the other.

SUBMITTER: Arfin SM

PROVIDER: S-EPMC41216 | biostudies-other | 1995 Aug

REPOSITORIES: biostudies-other

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Eukaryotic methionyl aminopeptidases: two classes of cobalt-dependent enzymes.

Arfin S M SM Kendall R L RL Hall L L Weaver L H LH Stewart A E AE Matthews B W BW Bradshaw R A RA

Proceedings of the National Academy of Sciences of the United States of America 19950801 17

Using partial amino acid sequence data derived from porcine methionyl aminopeptidase (MetAP; methionine aminopeptidase, peptidase M; EC 3.4.11.18), a full-length clone of the homologous human enzyme has been obtained. The cDNA sequence contains 2569 nt with a single open reading frame corresponding to a protein of 478 amino acids. The C-terminal portion representing the catalytic domain shows limited identity with MetAP sequences from various prokaryotes and yeast, while the N terminus is rich i ...[more]

PMID: 7644482

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Project description:β-Aminopeptidases have the unique capability to hydrolyze N-terminal β-amino acids, with varied preferences for the nature of β-amino acid side chains. This unique capability makes them useful as biocatalysts for synthesis of β-peptides and to kinetically resolve β-peptides and amides for the production of enantiopure β-amino acids. To date, six β-aminopeptidases have been discovered and functionally characterized, five from Gram-negative bacteria and one from a fungus, Aspergillus Here we report on the purification and characterization of an additional four β-aminopeptidases, one from a Gram-positive bacterium, Mycolicibacterium smegmatis (BapAMs), one from a yeast, Yarrowia lipolytica (BapAYlip), and two from Gram-negative bacteria isolated from activated sludge identified as Burkholderia spp. (BapABcA5 and BapABcC1). The genes encoding β-aminopeptidases were cloned, expressed in Escherichia coli, and purified. The β-aminopeptidases were produced as inactive preproteins that underwent self-cleavage to form active enzymes comprised of two different subunits. The subunits, designated α and β, appeared to be tightly associated, as the active enzyme was recovered after immobilized-metal affinity chromatography (IMAC) purification, even though only the α-subunit was 6-histidine tagged. The enzymes were shown to hydrolyze chromogenic substrates with the N-terminal l-configurations β-homo-Gly (βhGly) and β3-homo-Leu (β3hLeu) with high activities. These enzymes displayed higher activity with H-βhGly-p-nitroanilide (H-βhGly-pNA) than previously characterized enzymes from other microorganisms. These data indicate that the new β-aminopeptidases are fully functional, adding to the toolbox of enzymes that could be used to produce β-peptides. Overexpression studies in Pseudomonas aeruginosa also showed that the β-aminopeptidases may play a role in some cellular functions.IMPORTANCE β-Aminopeptidases are unique enzymes found in a diverse range of microorganisms that can utilize synthetic β-peptides as a sole carbon source. Six β-aminopeptidases have been previously characterized with preferences for different β-amino acid substrates and have demonstrated the capability to catalyze not only the degradation of synthetic β-peptides but also the synthesis of short β-peptides. Identification of other β-aminopeptidases adds to this toolbox of enzymes with differing β-amino acid substrate preferences and kinetics. These enzymes have the potential to be utilized in the sustainable manufacture of β-amino acid derivatives and β-peptides for use in biomedical and biomaterial applications. This is important, because β-amino acids and β-peptides confer increased proteolytic resistance to bioactive compounds and form novel structures as well as structures similar to α-peptides. The discovery of new enzymes will also provide insight into the biological importance of these enzymes in nature.

Project description:This paper compares the biophysical and mechanistic properties of a typical type I dehydroquinase (DHQase), from the biosynthetic shikimate pathway of Escherichia coli, and a typical type II DHQase, from the quinate pathway of Aspergillus nidulans. C.d. shows that the two proteins have different secondary-structure compositions; the type I enzyme contains approx. 50% alpha-helix while the type II enzyme contains approx. 75% alpha-helix. The stability of the two types of DHQase was compared by denaturant-induced unfolding, as monitored by c.d., and by differential scanning calorimetry. The type II enzyme unfolds at concentrations of denaturant 4-fold greater than the type I and through a series of discrete transitions, while the type I enzyme unfolds in a single transition. These differences in conformational stability were also evident from the calorimetric experiments which show that type I DHQase unfolds as a single co-operative dimer at 57 degrees C whereas the type II enzyme unfolds above 82 degrees C and through a series of transitions suggesting higher orders of structure than that seen for the type I enzyme. Sedimentation and Mr analysis of both proteins by analytical ultracentrifugation is consistent with the unfolding data. The type I DHQase exists predominantly as a dimer with Mr = 46,000 +/- 2000 (a weighted average affected by the presence of monomer) and has a sedimentation coefficient s0(20,w) = 4.12 (+/- 0.08) S whereas the type II enzyme is a dodecamer, weight-average Mr = 190,000 +/- 10,000 and has a sedimentation coefficient, s0(20,w) = 9.96 (+/- 0.21) S. Although both enzymes have reactive histidine residues in the active site and can be inactivated by diethyl pyrocarbonate, the possibility that these structurally dissimilar enzymes catalyse the same dehydration reaction by the same catalytic mechanism is deemed unlikely by three criteria: (1) they have very different pH/log kcat. profiles and pH optima; (2) imine intermediates, which are known to play a central role in the mechanism of type I enzymes, could not be detected (by borohydride reduction) in the type II enzyme; (3) unlike Schiff's base-forming type I enzymes, there are no conserved lysine residues in type II amino acid sequences.

Dataset Information

Eukaryotic methionyl aminopeptidases: two classes of cobalt-dependent enzymes.

Publications

Eukaryotic methionyl aminopeptidases: two classes of cobalt-dependent enzymes.

Similar Datasets

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