Tài liệu Báo cáo khoa học: Destabilization of psychrotrophic RNase HI in a localized fashion as

Destabilization of psychrotrophic RNase HI in a localized

fashion as revealed by mutational and X-ray

crystallographic analyses

Muhammad S. Rohman1

, Takashi Tadokoro1

, Clement Angkawidjaja1

, Yumi Abe1

,

Hiroyoshi Matsumura2,3, Yuichi Koga1

, Kazufumi Takano1,3 and Shigenori Kanaya1

1 Department of Material and Life Science, Graduate School of Engineering, Osaka University, Japan

2 Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Japan

3 CREST, JST, Osaka, Japan

Psychrophiles and psychrotrophs are defined as micro￾organisms that can grow even at around 0 C [1].

Enzymes from these microorganisms are usually less

stable than those from mesophiles and thermophiles

[2–4]. It has been reported that a decreased number of

ion pairs and hydrogen bonds, decreased hydrophobic

interactions and packing at the core, an increased

fraction of nonpolar surface area, a decreased surface

hydrophilicity, decreased helix stability and a

decreased number of proline residues in the loop

regions are responsible for their thermolability [5–8].

However, the destabilization mechanism of these

enzymes remains to be fully understood. One promis￾ing strategy to understand this mechanism is to

Keywords

crystal structure; destabilization mechanism;

RNase HI; Shewanella oneidensis MR-1;

thermostabilizing mutations

Correspondence

S. Kanaya, Department of Material and Life

Science, Graduate School of Engineering,

Osaka University, 2-1, Yamadaoka, Suita,

Osaka 565-0871, Japan

Fax: +81 6 6879 7938

Tel: +81 6 6879 7938

E-mail: [email protected]

(Received 26 September 2008, revised 11

November 2008, accepted 19 November

2008)

doi:10.1111/j.1742-4658.2008.06811.x

The Arg97 fi Gly and Asp136 fi His mutations stabilized So-RNase HI

from the psychrotrophic bacterium Shewanella oneidensis MR-1 by 5.4

and 9.7 C, respectively, in Tm, and 3.5 and 6.1 kJÆmol)1

, respectively, in

DG(H2O). These mutations also stabilized the So-RNase HI derivative

(4·-RNase HI) with quadruple thermostabilizing mutations in an additive

manner. As a result, the resultant sextuple mutant protein (6·-RNase HI)

was more stable than the wild-type protein by 28.8 C in Tm and 27.0

kJÆmol)1 in DG(H2O). To analyse the effects of the mutations on the pro￾tein structure, the crystal structure of the 6·-RNase HI protein was deter￾mined at 2.5 A˚ resolution. The main chain fold and interactions of the

side-chains of the 6·-RNase HI protein were basically identical to those of

the wild-type protein, except for the mutation sites. These results indicate

that all six mutations independently affect the protein structure, and are

consistent with the fact that the thermostabilizing effects of the mutations

are roughly additive. The introduction of favourable interactions and the

elimination of unfavourable interactions by the mutations contribute to the

stabilization of the 6·-RNase HI protein. We propose that So-RNase HI is

destabilized when compared with its mesophilic and thermophilic coun￾terparts in a localized fashion by increasing the number of amino acid

residues unfavourable for protein stability.

Abbreviations

4·-RNase HI, So-RNase HI derivative with Asn29 fi Lys, Asp39 fi Gly, Met76 fi Val and Lys90 fi Asn mutations; 5·-RNase HI, 4·-RNase

HI derivative with additional Arg97 fi Gly mutation; 6·-RNase HI, 5·-RNase HI derivative with additional Asp136 fi His mutation; D136H￾RNase HI, So-RNase HI derivative with Asp136 fi His mutation; Ec-RNase HI, E. coli RNase HI; GdnHCl, guanidine hydrochloride; PDB,

Protein Data Bank; R97G-RNase HI, So-RNase HI derivative with Arg97 fi Gly mutation; So-RNase HI, RNase HI from

Shewanella oneidensis MR-1; Tt-RNase HI, RNase HI from Thermus thermophilus.

FEBS Journal 276 (2009) 603–613 ª 2008 The Authors Journal compilation ª 2008 FEBS 603