Tài liệu Báo cáo Y học: Characterization of a cloned subtilisin-like serine proteinase from a

Characterization of a cloned subtilisin-like serine proteinase

from a psychrotrophic Vibrio species

Jo´ hanna Arno´rsdo´ttir1,2, Ru´ na B. Sma´rado´ttir1

, O´ lafur Th. Magnu´ sson2

, Sigrı´dur H. Thorbjarnardo´ttir1

,

Gudmundur Eggertsson1 and Magnu´ s M. Kristja´ nsson2

1

Institute of Biology, University of Iceland; and 2

Department of Biochemistry, Science Institute, University of Iceland,

Reykjavik, Iceland

The gene encoding a subtilisin-like serine proteinase in the

psychrotrophic Vibrio sp. PA44 has been successfully

cloned, sequenced and expressed in Escherichia coli. The

gene is 1593 basepairs and encodes a precursor protein of

530 amino acid residues with a calculated molecular mass

of 55.7 kDa. The enzyme is isolated, however, as an active

40.6-kDa proteinase, without a 139amino acid residue

N-terminal prosequence. Under mild conditions the

enzyme undergoes a further autocatalytic cleavage to give

a 29.7-kDa proteinase that retains full enzymatic activity.

The deduced amino acid sequence of the enzyme has high

homology to proteinases of the proteinase K family of

subtilisin-like proteinases. With respect to the enzyme

characteristics compared in this study the properties of the

wild-type and recombinant proteinases are the same.

Sequence analysis revealed that especially with respect to

the thermophilic homologues, aqualysin I from Thermus

aquaticus and a proteinase from Thermus strain Rt41A,

the cold-adapted Vibrio-proteinase has a higher content of

polar/uncharged amino acids, as well as aspartate resi￾dues. The thermophilic enzymes had a higher content of

arginines, and relatively higher number of hydrophobic

amino acids and a higher aliphatic index. These factors

may contribute to the adaptation of these proteinases to

different temperature conditions.

Keywords: cold adaptation; psychrotrophic; Vibrio-protein￾ase; proteinase K-like; subtilisin-like proteinase.

Many microorganisms and ectothermic animals live under

environmental temperatures that fluctuate in the range )2

to 10
C without the opportunity to regulate their cellular

temperatures [1–3]. In fact, cold temperature is the most

widespread physiological stress condition that organisms

have either to adapt to or to avoid. Adaptive changes in

protein structure and function induced by cold are of prime

importance for cold acclimation and survival processes [4].

A common denominator of evolutionary adaptive changes

of proteins appears to be the conservation and optimization

of the functional state of the proteins, such that they are in

corresponding states with respect to functionally important

motions, under the different physical conditions to which

the proteins have adapted [5]. It has been suggested that in

order to maintain such
corresponding states for efficient

biological function at low temperatures, cold-adapted

proteins must have adopted a higher degree of conforma￾tional flexibility [5–11]. As such cold-adaptive strategies

would require weakening or alteration of some intramole￾cular interactions, the structural stability of cold-adapted

proteins is expected to be diminished in comparison

with their counterparts adapted to higher temperatures

[6–8,11,12]. This has indeed been generally observed for

naturally occurring psychrophilic enzymes studied to date.

Recent studies in which directed evolution was used to

induce cold adaptive properties in a mesophilic enzyme and

increased thermal stability in a psychrophilic enzyme have,

however, indicated that there may not be a strict correlation

between increased activity at low temperatures and

decreased thermostability [13–15].

In recent years, there has been a growing interest in

enzymes from psychrophilic microorganisms, both as

models in studies on thermal stability and molecular

adaptation of proteins, as well as potential candidates for

biotechnological applications. Several enzymes from psy￾chrophilic bacteria have now been characterized [16–35] and

crystal structures of citrate synthase [23], triose-phosphate

isomerase [24], a-amylase [27,28], and that of malate

dehydrogenase [31] have been published. The psychrophilic

enzymes characterized so far generally have higher catalytic

activities at low temperatures and are less thermostable than

their counterparts from mesophiles. Comparative studies

where available crystal structures, sequences or three￾dimensional homology models of psychrophilic proteins

have been compared with homologous meso- and/or

thermophilic proteins have shown that a general set of rules

does not seem to exist for cold adaptation of proteins. Cold￾adaptive mechanisms seem to involve weakening of certain

Correspondence to M. M. Kristja´nsson, Department of Biochemistry,

Science Institute, University of Iceland, Dunhaga 3, 107 Reykjavik,

Iceland. Fax: + 354 5528911, Tel.: + 354 5254800,

E-mail: [email protected]

Abbreviations: AQUI, aqualysin I; GdmSCN, guanidinium

thiocyanate; GdmCl, guanidinium chloride; PRK, proteinase K;

Suc-AAPF-NH-Np, succinyl-AlaAlaProPhe-p-nitroanilide;

VPR, Vibrio-proteinase.

Enzymes: aqualysin I (EC 3.4.21.-); proteinase K (EC 3.4.21.64);

Vibrio-proteinase (EC 3.4.21.-)

Note: the sequence reported in this paper has been deposited in the

GenBank database (accession number AF521587).

(Received 19June 2002, revised 11 September 2002,

accepted 16 September 2002)

Eur. J. Biochem. 269, 5536–5546 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03259.x