Engineering a novel specificity in subtilisin BPN'

Rheinnecker, Michael; Baker, Graham R.; Eder, Jörg; Fersht, Alan R.

doi:10.1021/bi00056a001

Cited by 53 publications

(61 citation statements)

References 32 publications

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“…Based on these structural studies, several groups have mutated the S 4 pocket in an attempt to alter the P 4 substrate specificity of subtilisin (21)(22)(23)(24)(25)(26). Wells and co-workers (27,28) found that substitutions of Asp for Tyr 104 in subtilisin BPNЈ increased cleavage of substrates containing a P 4 Arg, but the resulting mutant protease did not discriminate between Arg and Phe at this position.…”

mentioning

confidence: 99%

Plasticity of Extended Subsites Facilitates Divergent Substrate Recognition by Kex2 and Furin

Rozan

Krysan

Rockwell

et al. 2004

Journal of Biological Chemistry

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Yeast Kex2 and human furin are subtilisin-related proprotein convertases that function in the late secretory pathway and exhibit similar though distinguishable patterns of substrate recognition. Although both enzymes prefer Arg at P 1 and basic residues at P 2 , the two differ in recognition of P 4 and P 6 residues. To probe P 4 and P 6 recognition by Kex2p, furin-like substitutions were made in the putative S 4 and S 6 subsites of Kex2. T252D and Q283E mutations were introduced to increase the preference for Arg at P 4 and P 6 , respectively. Glu 255 was replaced with Ile to limit recognition of P 4 Arg. The effects of putative S 4 and S 6 mutations were determined by examining the cleavage by purified mutant enzymes of a series of fluorogenic substrates with systematic changes in P 4 and/or P 6 . Whereas wild type Kex2 exhibited little preference for Arg at P 6 , the T252D mutant and T252D/Q283E double mutant exhibited clear interactions with P 6 Arg. Moreover, the T252D and T252D/Q283E substitutions altered the influence of the P 6 residue on P 4 recognition. We infer that cross-talk between S 4 and S 6 , not seen in furin, allows wild type and mutant forms of Kex2 to adapt their subsites for altered modes of recognition. This apparent plasticity may allow the subsites to rearrange their local environment to interact with different substrates in a productive manner. E255I-Kex2 exhibited significantly decreased recognition of P 4 Arg in a tetrapeptide substrate with Lys at P 1 , although the general pattern of selectivity for aliphatic residues at P 4 remained unchanged.The subtilisin superfamily includes a subfamily of related processing proteases, the proprotein convertases that function in the late secretory pathway of diverse eukaryotic organisms including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and mammals (1-4). Unlike the degradative subtilisins, which display a broad substrate specificity for hydrophobic residues (5), the proprotein convertases are post-translational modifying enzymes that process secretory proteins in a sequence-specific manner. In general, these proteases cleave C-terminal to clusters of basic residues, but their exact sequence specificity differs among the members of this family, even though they are Ն45% identical within their subtilisin-related domains. Similarities and differences in substrate recognition were illustrated by the enzymatic characterization of two members of this family, the S. cerevisiae protease, Kex2, and the human homologue, furin.A detailed understanding of substrate recognition by Kex2 and furin has emerged from extensive analysis of the purified secreted, soluble enzymes using model peptide substrates. Based on these studies, the consensus cleavage site for Kex2 was determined to be (Ali/Arg)-Xaa-(Lys/Arg)-Arg2 (where Ali indicates an aliphatic amino acid), with the principal determinants being a basic residue at P 2 and Arg at P 1 (6 -9).

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mentioning

confidence: 99%

Plasticity of Extended Subsites Facilitates Divergent Substrate Recognition by Kex2 and Furin

Rozan

Krysan

Rockwell

et al. 2004

Journal of Biological Chemistry

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“…Like in mesentericopeptidase, a threonine residue is located at position 130 in subtilisin RT-5, in contrast to Ser 13° in all other subtilisins. This Ser 13° is involved in hydrogen-bonding with Tyr TM at the entrance of the P4-binding pocket [3]. Mutations to remove this H bond and to increase the size of the S4-binding pocket resulted in a mutant enzyme with increased catalytic efficiency for large hydrophobic residues at position P4 [3].…”

Section: Discussionmentioning

confidence: 99%

“…This Ser 13° is involved in hydrogen-bonding with Tyr TM at the entrance of the P4-binding pocket [3]. Mutations to remove this H bond and to increase the size of the S4-binding pocket resulted in a mutant enzyme with increased catalytic efficiency for large hydrophobic residues at position P4 [3]. The increased preference of subtilisin RT-5 toward hydrophobic residues like valine and phenylalanine at the P4 position may, therefore, correlate with the substitution of Ser 13° to Thr 13°.…”

Section: Discussionmentioning

confidence: 99%

“…Several Bacillus species produce industrially important extracellular enzymes, in particular the alkaline protease subtilisin, and a neutral metalloprotease, isolated from overproducing mutants [1] and used industrially for decades [2]. Many further forms have been analysed [3] and subtilisin BPN' [4], subtilisin Carlsberg [5], proteinase K [6], thermitase [7] and aqualysin I [8] are natural members of the subtilisin family with known structures. Thermitase, proteinase K and aqualysin I are more thermostable than the other subtilisins.…”

Section: Introductionmentioning

confidence: 99%

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Isolation, characterization and structure of subtilisin from a thermostable Bacillus subtilis isolate

et al. 1995

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A serine protease has been isolated and characterized from Bacillus subtilis, strain RT-5 (a thermostahle soil isolate from the Tharparkar desert of Pakistan) able to grow at 55 ° C. The primary structure was established by a combination of protein and DNA-sequence analyses. The amino-acid sequence, inhibition pattern and solubility properties identify the enzyme as a subtilisin. It has 43 amino-acid replacements toward subtilisin BPN' and as much as 83 replacements toward another subtilisin, confirming that strain variabilities are extensive between different subtilisin forms. However, the structure is identical to one of unknown functional properties deduced from DNA and is closely related to mesentericopeptidase hut that homologue is not thermostable. From comparisons with that form and with snbtilisin BPN', it is concluded that replacements of Ala-~ Ser at positions 85 and 89, Ser--->Ala at position 88 and Asp or Ser--~Asn at position 259 may promote thermostahility.

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“…Whereas the trypsin specifi city is mainly determined by the single large S1-binding site selective for Arg or Lys, the specifi city of the subtilisin is rather broad and is determined by the two S1-and S4-binding sites (Wright et al , 1969 ). Due to the wide industrial use of subtilisin, there were numerous attempts to modify its specifi city (Rheinnecker et al , 1993(Rheinnecker et al , , 1994Takagi et al , 1997 ).…”

Section: Inhibition Of the S8 Family Serine Proteasesmentioning

confidence: 99%

β-Trefoil inhibitors – from the work of Kunitz onward

Renko

Sabotič²,

Turk³

2012

Biological Chemistry

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Protein protease inhibitors are the tools of nature in controlling proteolytic enzymes. They come in different shapes and sizes. The β -trefoil protease inhibitors that come from plants, fi rst discovered by Kunitz, were later complemented with representatives from higher fungi. They inhibit serine (families S1 and S8) and cysteine proteases (families C1 and C13) as well as other hydrolases. Their versatility is the result of the plasticity of the loops coming out of the stable β -trefoil scaffold. For this reason, they display several different mechanisms of inhibition involving different positions of the loops and their combinations. Natural diversity, as well as the initial successes in de novo protein engineering, makes the β -trefoil proteins a promising starting point for the generation of strong, specifi c, multitarget inhibitors capable of inhibiting multiple types of hydrolytic enzymes and simultaneously interacting with different protein, carbohydrate, or DNA molecules. This pool of knowledge opens up new possibilities for the exploration of their naturally occurring as well as modifi ed properties for applications in many fi elds of medicine, biotechnology, and agriculture.

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Engineering a novel specificity in subtilisin BPN'

Cited by 53 publications

References 32 publications

Plasticity of Extended Subsites Facilitates Divergent Substrate Recognition by Kex2 and Furin

Plasticity of Extended Subsites Facilitates Divergent Substrate Recognition by Kex2 and Furin

Isolation, characterization and structure of subtilisin from a thermostable Bacillus subtilis isolate

β-Trefoil inhibitors – from the work of Kunitz onward

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