PDBsum entry 1sio

Hydrolase/hydrolase inhibitor

PDB id: 1sio

Contents

Protein chains

357 a.a. *

Ligands

ACE-ILE-PRO-PHL ×3

SO4 ×3

Metals

_CA ×3

Waters ×935

* Residue conservation analysis

PDB id:

1sio

Name:

Hydrolase/hydrolase inhibitor

Title:

Structure of kumamolisin-as complexed with a covalently-bound inhibitor, acipf

Structure:

Kumamolisin-as. Chain: a, b, c. Engineered: yes. Ace-ile-pro-phl peptide inhibitor. Chain: d, e, f. Engineered: yes

Source:

Alicyclobacillus sendaiensis. Organism_taxid: 192387. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008. Synthetic: yes. Other_details: the peptide was chemically synthesized.

Biol. unit:

Dimer (from PQS)

Resolution:

1.80Å R-factor: 0.173 R-free: 0.243

Authors:

M.Li,A.Wlodawer,A.Gustchina,N.Tsuruoka,M.Ashida,H.Minakata,H.Oyama, K.Oda,T.Nishino,T.Nakayama

Key ref:

A.Wlodawer et al. (2004). Crystallographic and biochemical investigations of kumamolisin-As, a serine-carboxyl peptidase with collagenase activity. J Biol Chem, 279, 21500-21510. PubMed id: 15014068 DOI: 10.1074/jbc.M401141200

Date:

01-Mar-04 Release date: 30-Mar-04

Protein chains

Q8GB88  (Q8GB88_9BACL) -  Kumamolisin-As from Alicyclobacillus sendaiensis

Seq: 553 a.a.

Struc: 357 a.a.

DOI no: 10.1074/jbc.M401141200

J Biol Chem 279:21500-21510 (2004)

PubMed id: 15014068

Crystallographic and biochemical investigations of kumamolisin-As, a serine-carboxyl peptidase with collagenase activity.

A.Wlodawer, M.Li, A.Gustchina, N.Tsuruoka, M.Ashida, H.Minakata, H.Oyama, K.Oda, T.Nishino, T.Nakayama.

ABSTRACT

Kumamolisin-As (previously called ScpA) is the first known example of a collagenase from the sedolisin family (MEROPS S53). This enzyme is active at low pH and in elevated temperatures. In this study that used x-ray crystallographic and biochemical methods, we investigated the structural basis of the preference of this enzyme for collagen and the importance of a glutamate residue in the unique catalytic triad (Ser(278)-Glu(78)-Asp(82)) for enzymatic activity. Crystal structures of the uninhibited enzyme and its complex with a covalently bound inhibitor, N-acetyl-isoleucyl-prolyl-phenylalaninal, showed the occurrence of a narrow S2 pocket and a groove that encompasses the active site and is rich in negative charges. Limited endoproteolysis studies of bovine type-I collagen as well as kinetic studies using peptide libraries randomized at P1 and P1', showed very strong preference for arginine at the P1 position, which correlated very well with the presence of a negatively charged residue in the S1 pocket of the enzyme. All of these features, together with those predicted through comparisons with fiddler crab collagenase, a serine peptidase, rationalize the enzyme's preference for collagen. A comparison of the Arrhenius plots of the activities of kumamolisin-As with either collagen or peptides as substrates suggests that collagen should be relaxed before proteolysis can occur. The E78H mutant, in which the catalytic triad was engineered to resemble that of subtilisin, showed only 0.01% activity of the wild-type enzyme, and its structure revealed that Ser(278), His(78), and Asp(82) do not interact with each other; thus, the canonical catalytic triad is disrupted.

Selected figure(s)

Figure 5.
FIG. 5. A superposition of the active sites of four proteases. Kumamolisin-As with bound AcIPF is shown in blue; sedolisin and bound tyrostatin are colored red and brown, respectively; subtilisin is shown in lilac, and its inhibitor eglin is shown in violet. Fiddler crab collagenase is colored green, whereas its inhibitor ecotin is orange. The C atoms of kumamolisin-As, sedolisin, and subtilisin were superimposed with the program ALIGN (34). Superposition of kumamolisin-As with fiddler crab collagenase is described under "Results."

Figure 6.
FIG. 6. Superposition of kumamolisin-As and fiddler crab collagenase. The two structures were superimposed as described under "Results." The main chain tracing of kumamolisin-As and fiddler crab collagenase is shown as red and blue ribbons, respectively, and the superimposed side chains of the active sites residues are shown as sticks in corresponding colors. The strands 127-132 in kumamolisin-As and 213-218 in collagenase are emphasized in black (see under "Results"). The inhibitor AcIPF is shown in gold, and a fragment of ecotin is shown in green.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 21500-21510) copyright 2004.

Figures were selected by the author.