Tài liệu Báo cáo khoa học: Crystal structure of a subtilisin-like serine proteinase from a

Crystal structure of a subtilisin-like serine proteinase from

a psychrotrophic Vibrio species reveals structural aspects

of cold adaptation

Jo´ hanna Arno´rsdo´ ttir1

, Magnu´ s M. Kristja´nsson2 and Ralf Ficner1

1 Abteilung fu¨r Molekulare Strukturbiologie, Institut fu¨r Mikrobiologie und Genetik, Georg-August Universita¨t Go¨ttingen, Germany

2 Department of Biochemistry, Science Institute, University of Iceland, Reykjavı´k, Iceland

Microorganisms inhabit the most diverse environments

on earth. Extremophiles are microorganisms that have

adapted to environmental conditions regarded by

humans as falling outside the normal range in terms of

temperature, pressure, salinity or pH. Extremophiles

have had to develop strategies to deal with environ￾mental stress, mainly by molecular adaptation of their

cell inventory. Of major importance in adapting to

extreme environmental conditions is the optimization

of protein function and stability. Enzymes from

extremophiles are essentially like their mesophilic

counterparts, sharing the same overall fold and

catalysing identical reactions via the same mechanisms,

while having adopted different traits regarding kinetic

and structural properties. Therefore, they provide

excellent tools for examining the molecular basis of

different protein properties, as well as the relation

between structure and function in enzymes. Regarding

temperature, organisms have been isolated from places

with temperatures as high as 113
C [1] and biological

activity has been detected in microbial samples at tem￾peratures as low as )20
C [2]. Thermo- and hyper￾thermophiles face the challenge of keeping their

macromolecules functional under the environmental

Keywords

cold adaptation; crystal structure;

psychrotrophic; subtilase; thermostability

Correspondence

R. Ficner, Abteilung fu¨r Molekulare

Strukturbiologie, Institut fu¨r Mikrobiologie

und Genetik, Universita¨t Go¨ttingen, Justus￾von-Liebig-Weg11, 37077 Go¨ttingen,

Germany

Fax: +49 551 391 4082

Tel: +49 551 391 4072

E-mail: [email protected]

Database

The coordinates and structure factors for

the final structure of Vibrio proteinase at

1.84 A˚ resolution have been deposited in

the Protein Data Bank under the accession

number 1SH7.

(Received 30 September 2004, revised 26

November 2004, accepted 9 December

2004)

doi:10.1111/j.1742-4658.2005.04523.x

The crystal structure of a subtilisin-like serine proteinase from the psychro￾trophic marine bacterium, Vibrio sp. PA-44, was solved by means of

molecular replacement and refined at 1.84 A˚ . This is the first structure of a

cold-adapted subtilase to be determined and its elucidation facilitates

examination of the molecular principles underlying temperature adaptation

in enzymes. The cold-adapted Vibrio proteinase was compared with known

three-dimensional structures of homologous enzymes of meso- and thermo￾philic origin, proteinase K and thermitase, to which it has high structural

resemblance. The main structural features emerging as plausible determi￾nants of temperature adaptation in the enzymes compared involve the char￾acter of their exposed and buried surfaces, which may be related to

temperature-dependent variation in the physical properties of water. Thus,

the hydrophobic effect is found to play a significant role in the structural

stability of the meso- and thermophile enzymes, whereas the cold-adapted

enzyme has more of its apolar surface exposed. In addition, the cold-adap￾ted Vibrio proteinase is distinguished from the more stable enzymes by its

strong anionic character arising from the high occurrence of uncompen￾sated negatively charged residues at its surface. Interestingly, both the cold￾adapted and thermophile proteinases differ from the mesophile enzyme in

having more extensive hydrogen- and ion pair interactions in their struc￾tures; this supports suggestions of a dual role of electrostatic interactions

in the adaptation of enzymes to both high and low temperatures. The

Vibrio proteinase has three calcium ions associated with its structure, one

of which is in a calcium-binding site not described in other subtilases.

832 FEBS Journal 272 (2005) 832–845 ª 2005 FEBS