Vladena Hlinková, Ľubica Urbániková and Jozef Ševčík
Institute of
Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, Bratislava
84 251, Slovakia,
RNases Sa and Sa2 are extracellular guanylspecific
ribonucleases produced by Streptomyces aureofaciens, strains BMK and
R8/26, respectively. Both enzymes specifically hydrolyse the phosphodiester
bond of RNA at the 3´-side of guanosine nucleotides [1, 2]. The tertiary
structure of the enzymes is almost identical and the amino-acids involved in
catalytic reaction are conserved. In spite of that, the kinetic parameters of the two
enzymes significantly differ, for instance catalytic efficiency of RNase Sa2 is
ten times lower than RNase Sa. To better understand the structure-function
relationships of RNase Sa2 we have solved structures of two crystal forms of
the complexes of RNase Sa2 with guanosine-3´-monophosphate (3´-GMP), the
product of the catalytic cleavage, and the structure of the complex of RNase
Sa2 with exo-guanosine-2´,3´-cyclophosphorothioate (2´,3´-GCPT), the
thio-analogue of the reaction intermediate. The exo-conformation
prevents it from cleaving by the enzyme. The structures
were solved with the program MOLREP [3] using RNase Sa2 with free active
site (Ševčík, unpublished results) as the search model and refined with the
program REFMAC5 [4] against
2.2Å data to final R-factors between 20-23%. The guanine base in the complexes
forms hydrogen bonds with the amide-group of Glu40, Asn41, Arg42 and
carboxy-group of Glu43. The base is also stabilised by
stacking interaction with the aromatic rings of Phe39, Tyr87 and Phe 90,
which form the bottom of the active site. The phosphate group forms hydrogen
bonds with Arg67, Arg71, His86 and Tyr87. The mononucleotides 3´-GMP and 2´,3´-GCPT are bound in the same way as in RNase
Sa [6, 7]. Comparison of the active sites of RNase Sa and Sa2 complexes with
3´-GMP and 2´,3´-GCPT showed that the only difference is in Gln32 of RNase Sa,
which is involved in binding of the phosphate group. In RNase Sa2 in this
position is Arg34, which does not bind the phosphate group because of its
unfavourable conformation. This may explain in part the differences in
enzymatic activity.
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2 Nazarov,V. Phd-Work. Institute Of Molecular Biology, Slovak Academy Of Sciences. (1991).
3 Vagin, A. & Teplyakov, A. J. Appl. Cryst. 30 (1997) 1022-1025.
4 Murshudov, G., Vagin, A. & Dodson, E. J. Acta Cryst. A53 (2001) 240-255.
6 Sevcik, J., Dodson, E.J. & Dodson G.G. Acta Cryst. B 47 (1991) 240-253.
7 Sevcik, J., Zegers, I., Wyns, L., Duter, Z. & Wilson, K. Eur. J. Biochem. 216 (1993) 301-305.