Crystal structure of subtilisin DY, a random mutant of subtilisin Carlsberg

Abstract

The crystal structure of subtilisin DY inhibited by N-benzyloxycarbonyl-Ala-Pro-Phe-chloromethyl ketone has been solved by molecular replacement with subtilisin Carlsberg as the starting model. The model has been refined to a crystallographic R factor (ϭ Σ | |F o | Ϫ |F c | | / Σ |F o |) of 15.1% using Xray diffraction data to 0.175 nm resolution. Subtilisin DY is an alkaline proteinase from the X-irradiated Japanese strain DY of Bacillus licheniformis, which normally produces subtilisin Carlsberg. It has very similar properties to subtilisin Carlsberg, with a slightly enhanced resistance to heat and guanidine hydrochloride-induced denaturation, in spite of the fact that the sequences of the two enzymes differ in 31 positions out of 274 residues. The close similarity in overall three-dimensional structure of subtilisins DY and Carlsberg and also their physicochemical properties, such as activity and stability, shows that nature aided by X-irradiation for rapid 'evolution' is able to accommodate considerable changes in sequence without substantial changes in property.Keywords : subtilisin; X-ray structure ; sequence variability; X-irradiation; mutagenesis.Subtilisins form a well-characterised family of extracellular alkaline serine proteinases, which have been found in various species of Bacillus [1], as well as in fungi and yeasts and even in higher eukaryotes, such as worms, insects and plants [2]. The increasing interest over the last decades in these enzymes has been due to the biological and industrial significance of their proteolytic activity. Since subtilisins have a very broad substrate specificity and are highly stable at neutral and alkaline pH, they are of considerable industrial importance as protein-degrading additives to detergents. Subtilisin DY is secreted by an X-ray irradiated strain of Bacillus licheniformis [12] and was isolated in a homogeneous state [13]. Sequence studies show high similarity of subtilisin DY to subtilisin Carlsberg [14], with 29 substitutions out of 274 residues (but see below). Comparing the physicochemical properties of the two enzymes, subtilisin DY is less susceptible to oxidation in the presence of hematoporphyrin [15] and shows slightly enhanced stability to heat and guanidine hydrochlorideinduced denaturation [16]. The melting temperature of subtilisin DY is 4°C higher than that of the wild-type Carlsberg. The kinetic parameters for the hydrolysis of succinyl-Ala 2 -Phe-methylcoumarylamide [17] and several peptidyl-4-nitroanilides [18] do not differ significantly from the kinetic constants for subtilisin Carlsberg. Co-crystallisation experiments with subtilisin DY and the proteinase inhibitor eglin-c [19] suggested that DY hydrolyses the peptide bond between residues 45 and 46 in the reactive centre of eglin-c, unlike other known subtilisins including Carlsberg.In this paper, we describe the 0.175-nm resolution analysis of the crystal structure of subtilisin DY in a 1:1 complex with the synthetic inhibitor N-benzyloxycarbonyl-Ala-Pro-Phe-chloromethyl ketone. For sub...

Network Analysis of Protein Structures Identifies Functional Residues

“…In subtilisin protease, 33 high closeness values identified both substrate binding and one of the two cation binding-sites, in addition to all catalytic site residues. While the substrate and catalytic sites are close to one another, the identified cation bindingsite is on the opposite side of the protein structure ( Figure 5).…”

A novel approach for enhancing the catalytic efficiency of a protease at low temperature: Reduction in substrate inhibition by chemical modification

“…2C). Structures of the savinase/ gramicidin S complex (1TK2) and structurally related subtilisin/inhibitor (1CSE, IBH6, 1LW6) (Bode et al, 1987;Eschenburg et al, 1998;Radisky and Koshland, 2002) and subtilisin/propeptide complexes (1SCJ) (Jain et al, 1998) show that the substrate specificity pockets are flanked by the loops connecting these two helices to the active site. Thus, the position of one of the azocasein lobes is likely to represent substrate binding, where the protein chain enters between these loops and binds to the active site.…”

Expression of Recombinant Plasmodium falciparumSubtilisin-like Protease-1 in Insect Cells

“…Fig. 7 shows a comparison of the PfSUB-1 primary sequence with the catalytic domains of four closely related bacterial subtilisins, all of the structures of which have been solved by x-ray crystallography (31)(32)(33)(34). The alignment also includes the deduced sequence of a P. yoelii gene very similar to pfsub-1, identified during BLAST searches of preliminary sequence data produced by The Institute for Genomic Research/Naval Medical Research Center P. yoelii genome sequencing project.…”