PDBsum entry 1mbq

Hydrolase

PDB id: 1mbq

Contents

Protein chain

220 a.a. *

Ligands

BEN

Metals

_CA

Waters ×91

* Residue conservation analysis

PDB id:

1mbq

Name:

Hydrolase

Title:

Anionic trypsin from pacific chum salmon

Structure:

Trypsin. Chain: a. Ec: 3.4.21.4

Source:

Oncorhynchus keta. Chum salmon. Organism_taxid: 8018. Organ: pyloric caeca

Resolution:

1.80Å R-factor: 0.166 R-free: 0.195

Authors:

E.Toyota,K.K.S.Ng,S.Kuninaga,H.Sekizaki,K.Itoh,K.Tanizawa,M.N.G.James

Key ref:

E.Toyota et al. (2002). Crystal structure and nucleotide sequence of an anionic trypsin from chum salmon (Oncorhynchus keta) in comparison with Atlantic salmon (Salmo salar) and bovine trypsin. J Mol Biol, 324, 391-397. PubMed id: 12445776 DOI: 10.1016/S0022-2836(02)01097-5

Date:

03-Aug-02 Release date: 11-Dec-02

Protein chain

Q8AV11  (Q8AV11_ONCKE) -  Anionic trypsin (Fragment) from Oncorhynchus keta

Seq: 222 a.a.

Struc: 220 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.4.21.4 - trypsin.
• Reaction: Preferential cleavage: Arg-|-Xaa, Lys-|-Xaa.

DOI no: 10.1016/S0022-2836(02)01097-5

J Mol Biol 324:391-397 (2002)

PubMed id: 12445776

Crystal structure and nucleotide sequence of an anionic trypsin from chum salmon (Oncorhynchus keta) in comparison with Atlantic salmon (Salmo salar) and bovine trypsin.

E.Toyota, K.K.Ng, S.Kuninaga, H.Sekizaki, K.Itoh, K.Tanizawa, M.N.James.

ABSTRACT

The nucleotide sequence and crystal structure of chum salmon trypsin (CST) are now reported. The cDNA isolated from the pyloric caeca of chum salmon encodes 222 amino acid residues, the same number of residues as the anionic Atlantic salmon trypsin (AST), but one residue less than bovine beta-trypsin (BT). The net charge on CST determined from the sum of all charged amino acid side-chains is -3. There are 79 sequence differences between CST and BT, but only seven sequence differences between CST and AST. Anionic CST isolated from pyloric caeca has also been purified and crystallized; the structure of the CST-benzamidine complex has been determined to 1.8A resolution. The overall tertiary structure of CST is similar to that of AST and BT, but some differences are observed among the three trypsins. The most striking difference is at the C terminus of CST, where the expected last two residues are absent. The absence of these residues likely increases the flexibility of CST by the loss of important interactions between the N and C-terminal domains. Similarly, the lack of Tyr151 in CST (when compared with BT) allows more space for Gln192 in the active site thereby increasing substrate accessibility to the binding pocket. Lys152 in CST also adopts the important role of stabilizing the loop from residue 142 to 153. These observations on CST provide a complementary view of a second cold-adapted trypsin, which in comparison with the structures of AST and BT, suggest a structural basis for differences in enzymatic activity between enzymes from cold-adapted species and mammals.

Selected figure(s)

Figure 1.
Figure 1. The overall tertiary structure of the CST-benzamidine complex. CST was co-crystallized with benzamidine by equilibrating droplets containing 0.62 mM CST, 30 mM MgSO[4], 0.6 mM CaCl[2], 6.0 mM benzamidine, 50 mM Tris-HCl buffer (pH 8.15) against reservoir solutions containing 1.4 M MgSO[4] and 0.1 M Tris-HCl buffer (pH 8.5). A crystal adopting the form of a rectangular prism (0.15 mm×0.15 mm×0.3 mm) was obtained after four days. X-ray data were collected at room temperature on a DIP 2030H image plate detector (Mac Science Co., Ltd), using double-mirror focusing optics and Cu Ka radiation (l=1.5418 Å) generated by a Rigaku rotating anode generator RU-200BH operating at 45 kV and 75 mA. The data were processed and reduced with the programs Denzo & Scalepack.[22.] The structure of CST was determined by molecular replacement using AMoRe. [23.] Strong solutions were obtained from a rotation function (cc=0.38) and translation function (cc=0.65) calculated over the resolution range 20-4.0 Å using AST as the search model (PDB accession number 2TBS). Following rigid-body refinement (R-factor (20-3.0 Å)=0.28), the model was rebuilt using the program XFIT.[24.] Seven sequence differences with AST at positions 28, 125, 152, 153, 170, 235, and 244 and the deletion of two C-terminal residues were incorporated into the model of CST. Conventional crystallographic refinement was carried out using CNS. [25.] Water molecules were added to the model where the difference density exceeded 4s, and the peaks were within a reasonable distance of hydrogen-bonding partners on the enzyme (<3.3 Å). The statistics of the diffraction data and the final refinement cycle are listed in Table 1. Figure 1 and Figure 2 were prepared using MOLSCRIPT [26.] and Raster3D. [27.]

Figure 2.
Figure 2. Superposition of the specificity pocket and catalytic residues of CST and AST. The rmsd between the 24 atoms in the catalytic triad residues in CST and AST is 0.19 Å. The C^a backbone traces of CST and AST are drawn in blue and yellow, respectively. Oxygen atoms are colored red and nitrogen atoms blue.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2002, 324, 391-397) copyright 2002.