Crystal structure of the alkaline proteinase Savinase™ from Bacillus lentus at 1.4 Å resolution

Betzel, Christian; Klupsch, Silke; Papendorf, Gerlind; Hastrup, Sven; Branner, Sven; Wilson, K.S.

doi:10.1016/0022-2836(92)90662-4

Cited by 151 publications

(108 citation statements)

References 57 publications

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“…subtilisin BPN' (Hirono et al, 1984;McPhalen et al, 1985;McPhalen and James, 1988;Heinz et al, 1991;Takeuchi et al, 1991a,b), subtilisin Carlsberg (Bode et al, 1986;McPhalen and James, 1988), subtilisin BL (Godette et al, 1992), high-alkaline protease (Betzel et al, 1992;Sobek et al, 1992; Van der Laan et al, 1992;Lange et al, 1994), mesentericopeptidase (Dauter The pro-nisin part is fully modified, as indicated by the modified residues. Dha, dehydroalanine; Dhb, dehydrobutyrine; Ala-S-Ala, lanthionine; Abu-S-Ala, β-methyllanthionine.…”

Section: Introductionmentioning

confidence: 99%

Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin

Siezen¹,

Rollema²,

Kuipers³

et al. 1995

Protein Eng Des Sel

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Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 12-05-2018Protein Engineering vol.8 no.2 pp. [117][118][119][120][121][122][123][124][125] 1995 Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin ITo whom correspondence should be addressed A model is presented for the 3-D structure of the catalytic domain of the putative leader peptidase NisP of Lactococcus lactis, and the interaction with its specific substrate, the precursor of the lantibiotic nisin. This homology model is based on the crystal structures of subtilisin BPN' and thermitase in complex with the inhibitor eglin. Predictions are made of the general protein fold, inserted loops, Ca2+ binding sites, aromatic interactions and electrostatic interactions of NisP. Cleavage of the leader peptide from precursor nisin by NisP is the last step in maturation of nisin. A detailed prediction of the substrate binding site attempts to explain the basis of specificity of NisP for precursor nisin. Specific acidic residues in the SI sub site of the substrate binding region of NisP appear to be of particular importance for electrostatic interaction with the PI Arg residue of precursor nisin after which cleavage occurs. The hydrophobic S4 subsite of NisP may also contribute to substrate binding as it does in subtilisins. Predictions of enzyme-substrate interaction were tested by protein engineering of precursor nisin and determining susceptibility of mutant precursors to cleavage by NisP. An unusual property of NisP predicted from this catalytic domain model is a surface patch near the substrate binding region which is extremely rich in aromatic residues. It may be involved in binding to the cell membrane or to hydrophobic membrane proteins, or it may serve as the recognition and binding region for the modified, hydrophobic C-terminal segment of precursor nisin. Similar predictions for the tertiary structure and substrate binding are made for the highly homologous protein EpiP, the putative leader peptidase for the lantibiotic epidermin from Staphylococcus epidermidis, but EpiP lacks the aromatic patch. Based on these models, protein engineering can be employed not only to test the predicted enzyme-substrate interactions, but also to design lantibiotic leader peptidases with a desired specificity.

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Section: Introductionmentioning

confidence: 99%

Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin

Siezen¹,

Rollema²,

Kuipers³

et al. 1995

Protein Eng Des Sel

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“…It has also been suggested that the side chain of Tyrl04 is very flexible, such that it can adjust the size of the S4 pocket [9, 151. In this context, it is surprising that subtilisin 309, with a Val occupying position 104 [16], also exhibits strong preference for aromatic groups in P4 [13]. The prescnt study was carried out in order to investigate the influence of the residue occupying position 104 on P4 substrate specificity.…”

mentioning

confidence: 99%

Mutational replacements in subtilisin 309

Bech

Sørensen

Breddam

1992

European Journal of Biochemistry

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The previous notion that the amino acid side chain at position 104 of subtilisins is involved in the binding of the side chain at position P4 of the substrate has becn invcstigated. The amino acid residue Val104 in subtilisin 309 has been replaced by Ala, Arg, Asp, Phei Ser, Trp and Tyr by site-directed mutagenesis. It is shown that the P4 specificity of this enzyme is not determined solely by the amino acid residue occupying position 104, as the enzyme exhibits a marked preference for aromatic groups in P4, regardless of the nature of the position-104 residue. With hydrophilic amino acid residues at this position, no involvement is seen in binding of either hydrophobic or hydrophilic amino acid residues at position P4 of the substrates. The substrate with Asp in P4 is an exception, as the preference for this substrate is increased dramatically by introduction of an arginine residue at position 104 in the enzyme, presumably due to a substrate-induced conformational change. However, when position 104 is occupied by hydrophobic residues, it is highly involved in binding of hydrophobic amino acid residues, either by increasing the hydrophobicity of S4 or by determining the size of the pocket. The results suggest that the amino acid residue at position 104 is mobile such that it is positioned in the S4 binding site only when it can interact favourably with the substrate's side chain at position P4.According to their substrate specificities, the endopeptidases can be divided into several groups. A number of endopeptidases, e.g. trypsin [3], have a very high specificity for particular amino acid residues at position PI, while others are less specific. In some of these enzymes with broadcr substrate preference, a binding subsite rcmote from the scissile bond is of primary importance, e.g. the S2 subsites of a-lytic protease [4] and papain [5], and the S4 subsites of elastase [6] and the subtilisins [7]. X-ray crystallographic studies of subtilisins BPN' and Carlsberg complexed with various inhibitor proteins [8 -121, and extensive kinetic characterisation of two subtilisins, subtilisin 309 from Bacillus lentils and subtilisin BPN' from Bacillus amyloliquefuciens [I 31, suggest that interaction between the substrate and the S4 binding pocket is at least as important for substrate preference as interaction in the S1 region.Subtilisin BPN' exhibits a marked preferencc for aromatic groups in P4 [13]; this has been claimed to be due to a tyrosine Enzyme. Subtilisin 309, a protease from Bacillus lentus (EC 3.4.21.14).Note. The binding-site notation is that of Schechter and Berger [I]. Accordingly, amino acid residues in the suhstrate are referred to as PI, Pz, . . . Pi and P;, P;, . . . Pi away from the scissile bond towards the N-and C-terminal amino acid residues of the substrate, respectively. Enzymc subsites are denoted S, , Sz, ... Si and S;, S;, ... SI in correspondcnce with the substrate. The numbering of amino acid residues is that of subtilisin BPN' from Bacillus miyloliquefaciens (21. residue, TyrlO4 located...

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“…They are, however, structurally and evolutionarily distinct (Smith et al , 1966 ;Wright et al , 1969 ;Betzel et al , 1992 ). The most important difference in the active site between the two families is in the binding site specifi city.…”

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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Crystal structure of the alkaline proteinase Savinase™ from Bacillus lentus at 1.4 Å resolution

Cited by 151 publications

References 57 publications

Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin

Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin

Mutational replacements in subtilisin 309

β-Trefoil inhibitors – from the work of Kunitz onward

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