81NISR RESEARCH GRANT
2004 Research Grant

Adaptation to Low-temperature of a Psychrotrophic Bacterium
Kazufumi TAKANO
Department of Material and Life Science, Osaka University
Research aims
We have previously shown that the cellular content ofFKBP22 (SIB1 FKBP22) from a psychrotrophic bacterium
Shewanella sp. SIB1 increases at 4°C, as compared to thatat 20°C.1 SIB1 FKBP22 exists as a homodimer and exhibits
the PPIase (Peptidyl Prolyl cis-trans Isomerase) activity
like other MIP-like FKBP subfamily proteins. However, theoptimum temperature of this protein for activity (10°C) ismuch lower than that of E. coli FKBP22 (25°C). We proposed that this activity facilitates efficient folding of proteins containing cis prolines in psychrotrophic bacteria atlow temperatures.
In this work, the N- and C-domains of SIB1 FKBP22
were overproduced in E. coli and purified in an amount sufficient for physicochemical studies. By comparing their activities and stabilities with those of the intact protein, we
showed that the unfolding temperature of SIB1 FKBP22 ismuch higher than the optimum temperature for activity.
Based on these results, we discuss a role of each domain ofSIB1 FKBP22 and a cold-adaptation mechanism of thisprotein.2Results
PPIase Activity. When the PPIase activity was determined at 10°C by protease coupling assay using ALPF (Nsuccinyl-Ala-Leu-Pro-Phe-p-nitroanilide) as a substrate, Cdomain exhibited the activity. The catalytic efficiency(kcat /Km) of C-domain was estimated to be 1.43 m M1 s1,which was 1.6 times higher than that of SIB1 FKBP22. The
temperature dependence of the PPIase activity of C-domain
was nearly identical to that of SIB1 FKBP22. In contrast,
when the PPIase activity was determined by RNase T1 refolding assay, C-domain exhibited much less activity ascompared to that of SIB1 FKBP22.
Thermal stability. Heat induced unfolding of N-domain, C-domain, and SIB1 FKBP22 were analyzed by DSC(differential scanning calorimetry). The DSC curves of Ndomain and C-domain showed single peaks at 44.7°C and35.6°C, respectively. In contrast, the DSC curve of SIB1

FKBP22 showed two distinct peaks at 32.5°C and 46.4°C.
These results indicate that the thermal unfolding transitionsof SIB1 FKBP22 at lower and higher temperatures represent those of its C-domain and N-domain, respectively.
Discussion
Thermal unfolding of C-domain is also initiated at
25°C. Nevertheless, SIB1 FKBP22 and C-domain both
exhibit the maximal PPIase activity at 10°C and their activities are greatly reduced at 20°C. These results suggest that
subtle conformational change around the active-site causes
great reduction of the enzymatic activity. The large difference in the temperatures for enzymatic inactivation andstructural unfolding has been observed for cold-adapted a amylase and family 8 xylanase from an Antarctic bacterium. The apparent optimal temperatures of these proteinsfor enzymatic activities are much lower than the temperatures at which any significant conformational event occurs.
In contrast, the optimal temperatures for the activities of
their mesophilic and thermophilic counterparts closely correlate with the temperatures for their structural transitions.
Thus, the large difference in the temperatures for enzymatic
inactivation and structural unfolding seems to be a characteristic feature of cold-adapted enzymes. It has been proposed that this difference is caused by a cold-adaptation
strategy termed “localized flexibility”. Although an increase in flexibility around the active site increases kcat by
reducing the energy cost of conformational change during
the catalytic reaction, it should increase Km concomitantly.
By restricting the increase of flexibility within small areas,cold-adapted enzymes prevent unfavorable increase in Km.
References[ 1 ] Suzuki Y, Haruki M, Takano K, Morikawa M, Kanaya S. (2004) Possible involvement of an FKBP family member protein from a psychrotrophic bacterium Shewanella sp. SIB1 in cold-adaptation, Eur.
J. Biochem., 271, 1372–1381.
[ 2 ] Suzuki Y, Takano K, Kanaya S. (2005) Stabilities and activities of
the N- and C-domains of FKBP22 from a psychrotrophic bacterium
overproduced in Escherichia coli, FEBS J., 272, 632–642.