Purification and properties of a pepstatin-insensitive carboxyl proteinase from a Gram-negative bacterium

Oda, Kōhei; Sugitani, Masafumi; Fukuhara, Ken-ichi; Murao, Sawao

doi:10.1016/0304-4165(87)90055-9

Cited by 68 publications

(39 citation statements)

References 36 publications

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“…Although DCI is generally thought to be an irreversible inhibitor, some bona fide serine proteases do regain activity following DCI inactivation (22). Our clear finding that DFP inactivates CLN2p/TPP-I is somewhat unexpected given the reported insensitivity of TPP-I (6,8,17) and a bacterial pepstatin-insensitive protease (23) to this inhibitor. However, other studies have reported partial to complete inhibition of TPP-I activity with high concentrations of DFP (15,16).…”

Section: Fig 4 Ph Dependence Of Dfp Inactivation and Tpp-i Activitymentioning

confidence: 73%

The Human CLN2 Protein/Tripeptidyl-Peptidase I Is a Serine Protease That Autoactivates at Acidic pH

Lin¹,

Sohár²,

Lackland³

et al. 2001

Journal of Biological Chemistry

131

134

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Section: Fig 4 Ph Dependence Of Dfp Inactivation and Tpp-i Activitymentioning

confidence: 73%

The Human CLN2 Protein/Tripeptidyl-Peptidase I Is a Serine Protease That Autoactivates at Acidic pH

Lin¹,

Sohár²,

Lackland³

et al. 2001

Journal of Biological Chemistry

131

134

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“…However, ScpA was insensitive to specific inhibitors of aspartic proteinases, such as pepstatin and DAN. It was also insensitive to specific inhibitors of metalloproteinases (EDTA), cysteine proteinases (N-ethylmaleimide), and serine proteinases (PMSF) (it has been shown that some serine-carboxyl proteinases are also insensitive to PMSF [15,20,22]). For comparison, known collagenases are strongly inhibited by metal chelators, and the collagenolytic serine proteinases are inhibited by PMSF.…”

Section: Discussionmentioning

confidence: 99%

“…The analysis suggested that, in the E. coli transformant cells, the 57-kDa precursor of rScpA should undergo the two-step removal of the N-terminal portions of the precursor to produce the mature 37-kDa species. Primary-structure analysis of ScpA revealed that ScpA showed sequence similarities to the members of the S53 family of proteinases, i.e., kumamolysin (15), ScpP (22,23), ScpX (19,20), and CLN2 (32). Until recently, these S53 family members had been classified into the A7 family of aspartic peptidase, mainly on the basis of their optimum pH for activity (pH 3.0 to 4.5) and the results of chemical modification (25) as well as site-directed mutagenesis studies (5).…”

Section: Discussionmentioning

confidence: 99%

Collagenolytic Serine-Carboxyl Proteinase fromAlicyclobacillus sendaiensisStrain NTAP-1: Purification, Characterization, Gene Cloning, and Heterologous Expression

Tsuruoka

Nakayama

Ashida

et al. 2003

Appl Environ Microbiol

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Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest. Alicyclobacillus sendaiensis strain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides. Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA. Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined. On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala*Gly-Pro-Ile-Gly (k cat , 5.41 s ؊1 ; K m , 32 M) and Met-Gly-ProArg*Gly-Phe-Pro-Gly-Ser (k cat , 351 s ؊1 ; K m , 214 M), where the asterisks denote the scissile bonds. The cloned scpA gene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da. Heterologous expression of the scpA gene in the Escherichia coli cells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein. Sequencing of the scpA gene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results. Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined.Collagen is an insoluble structural protein that accounts for approximately 30% of the total weight of animal proteins and is produced in large quantities as a by-product in livestock industries. It has recently been shown that the enzymatic degradation of collagen as well as that of gelatin, a denatured form of collagen, allows efficient utilization of these structural proteins. This degradation produces peptides, the collagen peptides, which have been shown to have several biological activities of industrial and medical interest (27), leading to the establishment of a wide variety of applications, e.g., an immunotherapeutic agent (7, 9), a moisturizer for cosmetics, a preservative (2), and seasoning and dietary materials (4).Generally, the enzymatic degradation of collagen is not affected by ordinary digestive proteinases but requires the collagen-specific, Zn 2ϩ -dependent metalloproteinases (collagenases). Although microbial collagenases have been found in a wide variety of mesophilic bacterial strains (3), the industrialscale application of known bacterial collagenases for collagen peptide production has been hampered because of their insufficient stability. In 2000...

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“…In this study we have identified, isolated, and characterized a glutamic peptidase (TGP1) 4 from the secretome of Talaromyces emersonii. This thermophile secretes commercially valuable biopolymer-degrading enzymes, such as cellulases (17) and xylanases (18); however, no characterization of peptidases has previously been performed.…”

mentioning

confidence: 99%

Inhibition of a Secreted Glutamic Peptidase Prevents Growth of the Fungus Talaromyces emersonii

O’Donoghue

Mahon

Goetz

et al. 2008

Journal of Biological Chemistry

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The thermophilic filamentous fungus Talaromyces emersonii secretes a variety of hydrolytic enzymes that are of interest for processing of biomass into fuel. Many carbohydrases have been isolated and characterized from this fungus, but no studies had been performed on peptidases. In this study, two acid-acting endopeptidases were isolated and characterized from the culture filtrate of T. emersonii. One of these enzymes was identified as a member of the recently classified glutamic peptidase family and was subsequently named T. emersonii glutamic peptidase 1 (TGP1). The second enzyme was identified as an aspartyl peptidase (PEP1). TGP1 was cloned and sequenced and shown to exhibit 64 and 47% protein identity to peptidases from Aspergillus niger and Scytalidium lignocolum, respectively. Substrate profiling of 16 peptides determined that TGP1 has broad specificity with a preference for large residues in the P1 site, particularly Met, Gln, Phe, Lys, Glu, and small amino acids at P1 such as Ala, Gly, Ser, or Thr. This enzyme efficiently cleaves an internally quenched fluorescent substrate containing the zymogen activation sequence (k cat /K m ‫؍‬ 2 ؋ 10 5 M ؊1 s ؊1 ). Maximum hydrolysis occurs at pH 3.4 and 50°C. The reaction is strongly inhibited by a transition state peptide analog, TA1 (K i ‫؍‬ 1.5 nM), as well as a portion of the propeptide sequence, PT1 (K i ‫؍‬ 32 nM). Ex vivo studies show that hyphal extension of T. emersonii in complex media is unaffected by the aspartyl peptidase inhibitor pepstatin but is inhibited by TA1 and PT1. This study provides insight into the functional role of the glutamic peptidase TGP1 for growth of T. emersonii.As chemoheterotrophs, filamentous fungi secrete a variety of polymer-degrading hydrolases such as peptidases and carbohydrases that degrade organic material in the local environment to provide essential nutrients for the growing hyphae. Many fungi release organic acids to acidify the environment while secreting enzymes that are optimally active under these conditions. Traditionally, acid-acting fungal peptidases were assigned to the aspartic protease family and include aspergillopepsin from various Aspergillus species (1) and penicillopepsin from Penicillium species (2). These enzymes have two active-site aspartic acid residues and are strongly inhibited by pepstatin. Recently, two distinct groups of acid peptidases were identified that are insensitive to pepstatin and lack the catalytic motif observed in pepsintype peptidases. One group, "sedolisins" possess a fold similar to members of the subtilisin family but contain an active site triad of serine (S), glutamic acid (E), and aspartic acid (D) (3). They are sensitive to leupeptin and have homologs in bacteria (4), fungi (5), slime mold (6), and mammals (7). The second group of pepstatin-insensitive acid peptidases share a distinct set of structural, enzymatic, and physicochemical properties, and to date have only been identified in a select group of fungi (8). Members of this novel family possess a catalytic dyad consis...

show abstract

Purification and properties of a pepstatin-insensitive carboxyl proteinase from a Gram-negative bacterium

Cited by 68 publications

References 36 publications

The Human CLN2 Protein/Tripeptidyl-Peptidase I Is a Serine Protease That Autoactivates at Acidic pH

The Human CLN2 Protein/Tripeptidyl-Peptidase I Is a Serine Protease That Autoactivates at Acidic pH

Collagenolytic Serine-Carboxyl Proteinase fromAlicyclobacillus sendaiensisStrain NTAP-1: Purification, Characterization, Gene Cloning, and Heterologous Expression

Inhibition of a Secreted Glutamic Peptidase Prevents Growth of the Fungus Talaromyces emersonii

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