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<meta content="Ma, Y.F." name="eprints.creators_name" />
<meta content="Evans, D.E" name="eprints.creators_name" />
<meta content="Logue, S.J." name="eprints.creators_name" />
<meta content="Langridge, P." name="eprints.creators_name" />
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<meta content="Mutations of barley β-amylase that imporve substrate-binding affinity
and thermostability" name="eprints.title" />
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<meta content="Mutation' fJ-Amylase . Barley' Thermostability . Substrate-binding affinity" name="eprints.keywords" />
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<meta content="Abstract Three allelic forms of barley fJ-amylase (Sd I, Sd2H and Sd2L) exhibit different thermostability and kinetic properties. These differences critically influence the malting quality of barley varieties. To understand the molecular basis for the different properties of these three allelic forms, Sd I and Sd2L f3-amylase cDNAs were cloned, and the effects of the amino acid substitutions between them were evaluated by site-directed mutagenesis.
The results showed that an R 115C mutation is responsible
for the difference in kinetic properties. This substitution resulted in an additional hydrogen bond which may create a more favourable environment for substrate-binding. The different thermostabilities of the f3-amylase forms are due to two amino acid substitutions (V233A and L347S), which increased the enzyme's thermostability
index T50 by 1.9°C and 2.1°C, respectively. The increased thermostability associated with these two mutations may be due to relief of steric strain and the interaction of the protein surface with solvent water. Although both V233A and L347S mutations increased thermostability, they affected the thermostability in different
ways. The replacement of L347 by serine seems to increase the thermostability by slowing thermal unfolding
of the protein during heating, while the replacement ofV233 by alanine appears to cause an acceleration ofthe refolding after heating. Because the different fJ-amylase properties determined by the three mutations (RI15C, V233A and L347S) are associated with malting quality of
barley variety, a mutant with high thermostability and substrate-binding affinity was generated by combining the three preferred amino acid residues C 115, A233 and S347 together. A possible approach to producing barley varieties with better malting quality by genetic engineering
is discussed." name="eprints.abstract" />
<meta content="2001-11" name="eprints.date" />
<meta content="published" name="eprints.date_type" />
<meta content="Molecular and General Genetics" name="eprints.publication" />
<meta content="266" name="eprints.volume" />
<meta content="3" name="eprints.number" />
<meta content="345-352" name="eprints.pagerange" />
<meta content="10.1007/s004380100566" name="eprints.id_number" />
<meta content="TRUE" name="eprints.refereed" />
<meta content="1617-4615" name="eprints.issn" />
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<meta content="Eglinton lK, Langridge P, Evans DE (1998) Therrnostability variation in alleles of barley beta-amylase. 1 Cereal Sci 28:301309
Erkkila Ml, Leah R, Ahokas H, Cameron-Mills V (1998) Alleledependent
barley grain fi-amylase activity. Plant Physiol 117:679-685
Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-Pdb viewer: an environment for comparative protein modelling. Electrophoresis 18:2714-2723 Hejgaard J, Boisen S (1980) High lysine proteins in Hiproly barley breeding: identification, nutritional significance and new screening methods. Hereditas 93:311-320 Kaneko T, Kihara M, Ito K (2000) Genetic analysis of fi-amylase thermostability to develop a DNA marker for malt fermentability
improvement in barley, Hordeum vulgare L. Plant Breeding 119:197-201 Kihara M, Kaneko T, Ito K (1998) Genetic variation of fi-amylase
thermostability among varieties of barley, Hordeum vulgare L., and relation to malting quality. Plant Breeding 117:425--428 Kihara M, Kaneko T, Ito K, Aida Y, Takeda K (1999) Geographical
variation of fi-amylase thermostability among varieties
of barley (Hordeum vulgare) and fi-amylase deficiency. Plant Breeding I 18:453--455 Kreis M, Williamson M, Buxton B, Pywell 1, Hejgaard 1, Svendsen I (1987) Primary structure and differential expression of fi-amyJase
in normal and mutant barleys. Eur 1 Biochem 169:517525
Laederach A, Dowd MK, Coutinho PM, Reilly Pl (1999) Automated
docking of maltose, 2-deoxymaltose, and maltotetraose into the soybean fi-amylase active site. Proteins 37:166-175 Ma Y-F, Stewart DC, Eglinton lK, Logue SJ, Langridge P, Evans DE (2000a) Comparative enzyme kinetics of two allelic forms of barley (Hordeum vulgare L.) beta-amylase. 1 Cereal Sci 31:335344
Ma Y-F, Egfinton lK, Evans DE, Logue Sl, Langridge P (2000b) Removal of the four C-terminal glycine-rich repeats enhances the thermostability of barley fi-amylase. Biochemistry
39: 13350-13355
MacGregor AW, LaBerge DE, Meredith OS (1971) Changes in barley kernels during growth and maturation. Cereal Chern 48:255-269
MacGregor AW, Bazin SL, Macri LJ, Babb lC (1999) Modelling the contribution of alpha-amylase, beta-amylase and limit dextrinase to starch degradation during mashing. 1 Cereal Sci 29:161-169
Mikami B, Hehre El, Sato M, Katsube Y, Hirose M, Morita Y, Sacchettini JC (1993) The 2.0-;\ resolution structure of soybean fi-amylase complexed with «-cyclodextrin. Biochemistry 32:6836-6845 Mikami B, Degano M, Hehre El, Sacchettini lC (1994) Crystal structure of soybean fi-amylase reacted with fi-maltose and maltal: active site components and their apparent roles in catalysis. Biochemistry 33:7779-7787 Mikami B, YOOI) Hye-Jin, Yoshigi N (1999) The crystal structure of the sevenfold mutant of barley fi-amylase with increased thermostability at 2.5 A resolution. 1 Mol BioI 285: 1235-1243
Nelson N (1944) Photometric adaptation of the Somogyi method for the determination of glucose. 1 BioI Chern 153:375-380 Peitsch MC (1995) Protein modelling by E-mail. Biotechnology
13:658--660
Peitsch MC (1996) ProMod and Swiss-Model: Internet-based tools for automated comparative protein modelling. Biochem Soc Trans 24:274-279
Perella FW (1988) EZ-FIT: a practical curve-fitting microcomputer program for the analysis of enzyme kinetic data on IBM-PC compatible computers. Anal Biochem 174:437--447
Stenholm K, Home S (1999) A new approach to limit dextrinase and its role in mashing. 1 Inst Brew 105:205-210
352
Takeda Y, Guan HP, Preiss J (1993) Branching of amylose by the branching isoenzymes of maize endosperm. Carbohydr Res 240:253-263
Totsuka A, Fukazawa C (1993) Expression and mutation of soybean
fi-amylase in Escherichia coli. Eur J Biochem 21:787-794
Wan Y, Lemaux PG (1994) Generation of large numbers of independently
transformed fertile barley plants. Plant Physiol 104:37-48
Yoshigi N, Okada Y, Sahara H, Koshino S (1994) PCR cloning and sequencing of the fi-amylase cDNA from barley. J Biochem 115:47-51
Yoshigi N, Okada Y, Maeba H, Sahara H, Tamaki T (1995) Construction of a plasmid used for the expression of a sevenfold-
mutant barley fi-amylase with increased thermostability in Escherichia coli and properties of the sevenfold-mutant fi-amylase.
J Biochem 118:562-567" name="eprints.referencetext" />
<meta content="Ma, Y.F. and Evans, D.E and Logue, S.J. and Langridge, P. (2001) Mutations of barley β-amylase that imporve substrate-binding affinity and thermostability. Molecular and General Genetics, 266 (3). pp. 345-352. ISSN 1617-4615" name="eprints.citation" />
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<meta content="Evans, D.E" name="DC.creator" />
<meta content="Logue, S.J." name="DC.creator" />
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<meta content="Abstract Three allelic forms of barley fJ-amylase (Sd I, Sd2H and Sd2L) exhibit different thermostability and kinetic properties. These differences critically influence the malting quality of barley varieties. To understand the molecular basis for the different properties of these three allelic forms, Sd I and Sd2L f3-amylase cDNAs were cloned, and the effects of the amino acid substitutions between them were evaluated by site-directed mutagenesis.
The results showed that an R 115C mutation is responsible
for the difference in kinetic properties. This substitution resulted in an additional hydrogen bond which may create a more favourable environment for substrate-binding. The different thermostabilities of the f3-amylase forms are due to two amino acid substitutions (V233A and L347S), which increased the enzyme's thermostability
index T50 by 1.9°C and 2.1°C, respectively. The increased thermostability associated with these two mutations may be due to relief of steric strain and the interaction of the protein surface with solvent water. Although both V233A and L347S mutations increased thermostability, they affected the thermostability in different
ways. The replacement of L347 by serine seems to increase the thermostability by slowing thermal unfolding
of the protein during heating, while the replacement ofV233 by alanine appears to cause an acceleration ofthe refolding after heating. Because the different fJ-amylase properties determined by the three mutations (RI15C, V233A and L347S) are associated with malting quality of
barley variety, a mutant with high thermostability and substrate-binding affinity was generated by combining the three preferred amino acid residues C 115, A233 and S347 together. A possible approach to producing barley varieties with better malting quality by genetic engineering
is discussed." name="DC.description" />
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<h1 class="ep_tm_pagetitle">Mutations of barley β-amylase that imporve substrate-binding affinity and thermostability</h1>
<p style="margin-bottom: 1em" class="not_ep_block"><span class="person_name">Ma, Y.F.</span> and <span class="person_name">Evans, D.E</span> and <span class="person_name">Logue, S.J.</span> and <span class="person_name">Langridge, P.</span> (2001) <xhtml:em>Mutations of barley β-amylase that imporve substrate-binding affinity and thermostability.</xhtml:em> Molecular and General Genetics, 266 (3). pp. 345-352. ISSN 1617-4615</p><p style="margin-bottom: 1em" class="not_ep_block"></p><table style="margin-bottom: 1em" class="not_ep_block"><tr><td valign="top" style="text-align:center"><a href="http://eprints.utas.edu.au/2334/1/Ma_et_al_2001_MGG.pdf"><img alt="[img]" src="http://eprints.utas.edu.au/style/images/fileicons/application_pdf.png" class="ep_doc_icon" border="0" /></a></td><td valign="top"><a href="http://eprints.utas.edu.au/2334/1/Ma_et_al_2001_MGG.pdf"><span class="ep_document_citation">PDF</span></a> - Full text restricted - Requires a PDF viewer<br />250Kb</td><td><form method="get" accept-charset="utf-8" action="http://eprints.utas.edu.au/cgi/request_doc"><input accept-charset="utf-8" value="2989" name="docid" type="hidden" /><div class=""><input value="Request a copy" name="_action_null" class="ep_form_action_button" onclick="return EPJS_button_pushed( '_action_null' )" type="submit" /> </div></form></td></tr></table><p style="margin-bottom: 1em" class="not_ep_block">Official URL: <a href="http://dx.doi.org/10.1007/s004380100566">http://dx.doi.org/10.1007/s004380100566</a></p><div class="not_ep_block"><h2>Abstract</h2><p style="padding-bottom: 16px; text-align: left; margin: 1em auto 0em auto">Abstract Three allelic forms of barley fJ-amylase (Sd I, Sd2H and Sd2L) exhibit different thermostability and kinetic properties. These differences critically influence the malting quality of barley varieties. To understand the molecular basis for the different properties of these three allelic forms, Sd I and Sd2L f3-amylase cDNAs were cloned, and the effects of the amino acid substitutions between them were evaluated by site-directed mutagenesis.
The results showed that an R 115C mutation is responsible
for the difference in kinetic properties. This substitution resulted in an additional hydrogen bond which may create a more favourable environment for substrate-binding. The different thermostabilities of the f3-amylase forms are due to two amino acid substitutions (V233A and L347S), which increased the enzyme's thermostability
index T50 by 1.9°C and 2.1°C, respectively. The increased thermostability associated with these two mutations may be due to relief of steric strain and the interaction of the protein surface with solvent water. Although both V233A and L347S mutations increased thermostability, they affected the thermostability in different
ways. The replacement of L347 by serine seems to increase the thermostability by slowing thermal unfolding
of the protein during heating, while the replacement ofV233 by alanine appears to cause an acceleration ofthe refolding after heating. Because the different fJ-amylase properties determined by the three mutations (RI15C, V233A and L347S) are associated with malting quality of
barley variety, a mutant with high thermostability and substrate-binding affinity was generated by combining the three preferred amino acid residues C 115, A233 and S347 together. A possible approach to producing barley varieties with better malting quality by genetic engineering
is discussed.</p></div><table style="margin-bottom: 1em" cellpadding="3" class="not_ep_block" border="0"><tr><th valign="top" class="ep_row">Item Type:</th><td valign="top" class="ep_row">Article</td></tr><tr><th valign="top" class="ep_row">Additional Information:</th><td valign="top" class="ep_row">The original publication is available at www.springerlink.com
</td></tr><tr><th valign="top" class="ep_row">Keywords:</th><td valign="top" class="ep_row">Mutation' fJ-Amylase . Barley' Thermostability . Substrate-binding affinity</td></tr><tr><th valign="top" class="ep_row">Subjects:</th><td valign="top" class="ep_row"><a href="http://eprints.utas.edu.au/view/subjects/300000.html">300000 Agricultural, Veterinary and Environmental Sciences</a></td></tr><tr><th valign="top" class="ep_row">ID Code:</th><td valign="top" class="ep_row">2334</td></tr><tr><th valign="top" class="ep_row">Deposited By:</th><td valign="top" class="ep_row"><span class="ep_name_citation"><span class="person_name">Dr Evan Evans</span></span></td></tr><tr><th valign="top" class="ep_row">Deposited On:</th><td valign="top" class="ep_row">31 Oct 2007 15:10</td></tr><tr><th valign="top" class="ep_row">Last Modified:</th><td valign="top" class="ep_row">09 Jan 2008 02:30</td></tr><tr><th valign="top" class="ep_row">ePrint Statistics:</th><td valign="top" class="ep_row"><a target="ePrintStats" href="/es/index.php?action=show_detail_eprint;id=2334;">View statistics for this ePrint</a></td></tr></table><p align="right">Repository Staff Only: <a href="http://eprints.utas.edu.au/cgi/users/home?screen=EPrint::View&eprintid=2334">item control page</a></p>
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