Crystal structures of the ribonuclease MC1 mutants N71T and N71S in complex with 5′-GMP: Structural basis for alterations in substrate specificity

Tomoyuki Numata, Akio Suzuki, Yoshimitsu Kakuta, Kazumi Kimura, Min Yao, Isao Tanaka, Yuichiro Yoshida, Tadashi Ueda, Makoto Kimura

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Ribonuclease MC1 (RNase MC1), isolated from bitter gourd seeds, is a uridine specific RNase belonging to the RNase T2 family. Mutations of Asn71 in RNase MC1 to the amino acids Thr (N71T) and Ser (N71 S) in guanosine preferential RNases altered the substrate specificity from uridine specific to guanosine specific, as shown by the transphosphorylation of diribonucleoside monophosphates [Numata, T., et al. (2001) Biochemistry 40, 524-530]. To elucidate the structural basis for the alteration of substrate specificity, crystal structures of the RNase MC1 mutants N71T and N71S, free or complexed with 5′-GMP, were determined at resolutions higher than 2 Å. In the N71T-5′-GMP and N71S-5′-GMP complexes, the guanine moiety was, as in the case of the uracil moiety bound to wild-type RNase MC1, firmly stabilized in the B2 site by an extensive network of hydrogen bonds and hydrophobic interactions. Structure comparisons showed that mutations of Asn71 to Thr or Ser cause an enlargement of the B2 site, which then make it feasible to insert a guanine base into the B2 site of mutants N71T and N71S. This binding further allows for hydrogen bonding interaction of the side chain hydroxyl groups of Thr71 or Ser71 with the N7 atom of the guanine base. The mode of guanine binding of mutants N71T and N71S was found to be essentially identical to that of a guanosine preferential RNase NW from Nicotiana glutinosa. In particular, hydrogen bonds between the N7 atom of the guanine base and the hydroxyl groups of the amino acids at position 71 (RNase MC1 numbering) were completely conserved in three guanosine preferential enzymes, thereby indicating that the hydrogen bond may play an essential role in guanine binding in guanosine preferential RNases in the RNase T2 family. Consequently, it can be concluded that amino acids at position 71 (RNase MC1 numbering) serve as one of the determinants for substrate specificity (or preference) in the RNase T2 fimily by changing the size and shape of the B2 site.

Original languageEnglish
Pages (from-to)5270-5278
Number of pages9
JournalBiochemistry
Volume42
Issue number18
DOIs
Publication statusPublished - May 13 2003

Fingerprint

Guanosine Monophosphate
ribonuclease T(2)
Guanine
Substrate Specificity
Guanosine
Crystal structure
Hydrogen bonds
Substrates
Ribonucleases
Hydrogen
Uridine
Amino Acids
Hydroxyl Radical
Momordica charantia
Atoms
Mutation
Biochemistry
Uracil
Hydrogen Bonding
Hydrophobic and Hydrophilic Interactions

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Crystal structures of the ribonuclease MC1 mutants N71T and N71S in complex with 5′-GMP : Structural basis for alterations in substrate specificity. / Numata, Tomoyuki; Suzuki, Akio; Kakuta, Yoshimitsu; Kimura, Kazumi; Yao, Min; Tanaka, Isao; Yoshida, Yuichiro; Ueda, Tadashi; Kimura, Makoto.

In: Biochemistry, Vol. 42, No. 18, 13.05.2003, p. 5270-5278.

Research output: Contribution to journalArticle

Numata, Tomoyuki ; Suzuki, Akio ; Kakuta, Yoshimitsu ; Kimura, Kazumi ; Yao, Min ; Tanaka, Isao ; Yoshida, Yuichiro ; Ueda, Tadashi ; Kimura, Makoto. / Crystal structures of the ribonuclease MC1 mutants N71T and N71S in complex with 5′-GMP : Structural basis for alterations in substrate specificity. In: Biochemistry. 2003 ; Vol. 42, No. 18. pp. 5270-5278.
@article{41b110d4afd44e02a90eef2d2b7c0c7b,
title = "Crystal structures of the ribonuclease MC1 mutants N71T and N71S in complex with 5′-GMP: Structural basis for alterations in substrate specificity",
abstract = "Ribonuclease MC1 (RNase MC1), isolated from bitter gourd seeds, is a uridine specific RNase belonging to the RNase T2 family. Mutations of Asn71 in RNase MC1 to the amino acids Thr (N71T) and Ser (N71 S) in guanosine preferential RNases altered the substrate specificity from uridine specific to guanosine specific, as shown by the transphosphorylation of diribonucleoside monophosphates [Numata, T., et al. (2001) Biochemistry 40, 524-530]. To elucidate the structural basis for the alteration of substrate specificity, crystal structures of the RNase MC1 mutants N71T and N71S, free or complexed with 5′-GMP, were determined at resolutions higher than 2 {\AA}. In the N71T-5′-GMP and N71S-5′-GMP complexes, the guanine moiety was, as in the case of the uracil moiety bound to wild-type RNase MC1, firmly stabilized in the B2 site by an extensive network of hydrogen bonds and hydrophobic interactions. Structure comparisons showed that mutations of Asn71 to Thr or Ser cause an enlargement of the B2 site, which then make it feasible to insert a guanine base into the B2 site of mutants N71T and N71S. This binding further allows for hydrogen bonding interaction of the side chain hydroxyl groups of Thr71 or Ser71 with the N7 atom of the guanine base. The mode of guanine binding of mutants N71T and N71S was found to be essentially identical to that of a guanosine preferential RNase NW from Nicotiana glutinosa. In particular, hydrogen bonds between the N7 atom of the guanine base and the hydroxyl groups of the amino acids at position 71 (RNase MC1 numbering) were completely conserved in three guanosine preferential enzymes, thereby indicating that the hydrogen bond may play an essential role in guanine binding in guanosine preferential RNases in the RNase T2 family. Consequently, it can be concluded that amino acids at position 71 (RNase MC1 numbering) serve as one of the determinants for substrate specificity (or preference) in the RNase T2 fimily by changing the size and shape of the B2 site.",
author = "Tomoyuki Numata and Akio Suzuki and Yoshimitsu Kakuta and Kazumi Kimura and Min Yao and Isao Tanaka and Yuichiro Yoshida and Tadashi Ueda and Makoto Kimura",
year = "2003",
month = "5",
day = "13",
doi = "10.1021/bi034103g",
language = "English",
volume = "42",
pages = "5270--5278",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "18",

}

TY - JOUR

T1 - Crystal structures of the ribonuclease MC1 mutants N71T and N71S in complex with 5′-GMP

T2 - Structural basis for alterations in substrate specificity

AU - Numata, Tomoyuki

AU - Suzuki, Akio

AU - Kakuta, Yoshimitsu

AU - Kimura, Kazumi

AU - Yao, Min

AU - Tanaka, Isao

AU - Yoshida, Yuichiro

AU - Ueda, Tadashi

AU - Kimura, Makoto

PY - 2003/5/13

Y1 - 2003/5/13

N2 - Ribonuclease MC1 (RNase MC1), isolated from bitter gourd seeds, is a uridine specific RNase belonging to the RNase T2 family. Mutations of Asn71 in RNase MC1 to the amino acids Thr (N71T) and Ser (N71 S) in guanosine preferential RNases altered the substrate specificity from uridine specific to guanosine specific, as shown by the transphosphorylation of diribonucleoside monophosphates [Numata, T., et al. (2001) Biochemistry 40, 524-530]. To elucidate the structural basis for the alteration of substrate specificity, crystal structures of the RNase MC1 mutants N71T and N71S, free or complexed with 5′-GMP, were determined at resolutions higher than 2 Å. In the N71T-5′-GMP and N71S-5′-GMP complexes, the guanine moiety was, as in the case of the uracil moiety bound to wild-type RNase MC1, firmly stabilized in the B2 site by an extensive network of hydrogen bonds and hydrophobic interactions. Structure comparisons showed that mutations of Asn71 to Thr or Ser cause an enlargement of the B2 site, which then make it feasible to insert a guanine base into the B2 site of mutants N71T and N71S. This binding further allows for hydrogen bonding interaction of the side chain hydroxyl groups of Thr71 or Ser71 with the N7 atom of the guanine base. The mode of guanine binding of mutants N71T and N71S was found to be essentially identical to that of a guanosine preferential RNase NW from Nicotiana glutinosa. In particular, hydrogen bonds between the N7 atom of the guanine base and the hydroxyl groups of the amino acids at position 71 (RNase MC1 numbering) were completely conserved in three guanosine preferential enzymes, thereby indicating that the hydrogen bond may play an essential role in guanine binding in guanosine preferential RNases in the RNase T2 family. Consequently, it can be concluded that amino acids at position 71 (RNase MC1 numbering) serve as one of the determinants for substrate specificity (or preference) in the RNase T2 fimily by changing the size and shape of the B2 site.

AB - Ribonuclease MC1 (RNase MC1), isolated from bitter gourd seeds, is a uridine specific RNase belonging to the RNase T2 family. Mutations of Asn71 in RNase MC1 to the amino acids Thr (N71T) and Ser (N71 S) in guanosine preferential RNases altered the substrate specificity from uridine specific to guanosine specific, as shown by the transphosphorylation of diribonucleoside monophosphates [Numata, T., et al. (2001) Biochemistry 40, 524-530]. To elucidate the structural basis for the alteration of substrate specificity, crystal structures of the RNase MC1 mutants N71T and N71S, free or complexed with 5′-GMP, were determined at resolutions higher than 2 Å. In the N71T-5′-GMP and N71S-5′-GMP complexes, the guanine moiety was, as in the case of the uracil moiety bound to wild-type RNase MC1, firmly stabilized in the B2 site by an extensive network of hydrogen bonds and hydrophobic interactions. Structure comparisons showed that mutations of Asn71 to Thr or Ser cause an enlargement of the B2 site, which then make it feasible to insert a guanine base into the B2 site of mutants N71T and N71S. This binding further allows for hydrogen bonding interaction of the side chain hydroxyl groups of Thr71 or Ser71 with the N7 atom of the guanine base. The mode of guanine binding of mutants N71T and N71S was found to be essentially identical to that of a guanosine preferential RNase NW from Nicotiana glutinosa. In particular, hydrogen bonds between the N7 atom of the guanine base and the hydroxyl groups of the amino acids at position 71 (RNase MC1 numbering) were completely conserved in three guanosine preferential enzymes, thereby indicating that the hydrogen bond may play an essential role in guanine binding in guanosine preferential RNases in the RNase T2 family. Consequently, it can be concluded that amino acids at position 71 (RNase MC1 numbering) serve as one of the determinants for substrate specificity (or preference) in the RNase T2 fimily by changing the size and shape of the B2 site.

UR - http://www.scopus.com/inward/record.url?scp=0037544149&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037544149&partnerID=8YFLogxK

U2 - 10.1021/bi034103g

DO - 10.1021/bi034103g

M3 - Article

C2 - 12731868

AN - SCOPUS:0037544149

VL - 42

SP - 5270

EP - 5278

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 18

ER -