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| - | [[Image:2d0k.gif|left|200px]] | + | {{Seed}} |
| | + | [[Image:2d0k.png|left|200px]] |
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| | {{STRUCTURE_2d0k| PDB=2d0k | SCENE= }} | | {{STRUCTURE_2d0k| PDB=2d0k | SCENE= }} |
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| - | '''Methionine-free mutant of Escherichia coli dihydrofolate reductase'''
| + | ===Methionine-free mutant of Escherichia coli dihydrofolate reductase=== |
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| - | ==Overview==
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| - | We developed a strategy for finding out the adapted variants of enzymes, and we applied it to an enzyme, dihydrofolate reductase (DHFR), in terms of its catalytic activity so that we successfully obtained several hyperactive cysteine- and methionine-free variants of DHFR in which all five methionyl and two cysteinyl residues were replaced by other amino acid residues. Among them, a variant (M1A/M16N/M20L/M42Y/C85A/M92F/C152S), named as ANLYF, has an approximately seven times higher k(cat) value than wild type DHFR. Enzyme kinetics and crystal structures of the variant were investigated for elucidating the mechanism of the hyperactivity. Steady-state and transient binding kinetics of the variant indicated that the kinetic scheme of the catalytic cycle of ANLYF was essentially the same as that of wild type, showing that the hyperactivity was brought about by an increase of the dissociation rate constants of tetrahydrofolate from the enzyme-NADPH-tetrahydrofolate ternary complex. The crystal structure of the variant, solved and refined to an R factor of 0.205 at 1.9-angstroms resolution, indicated that an increased structural flexibility of the variant and an increased size of the N-(p-aminobenzoyl)-L-glutamate binding cleft induced the increase of the dissociation constant. This was consistent with a large compressibility (volume fluctuation) of the variant. A comparison of folding kinetics between wild type and the variant showed that the folding of these two enzymes was similar to each other, suggesting that the activity enhancement of the enzyme can be attained without drastic changes of the folding mechanism.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_16510443}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 16510443 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_16510443}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Katayanagi, K.]] | | [[Category: Katayanagi, K.]] |
| | [[Category: Alpha + beta]] | | [[Category: Alpha + beta]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 23:28:31 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun Jul 27 17:37:32 2008'' |
Revision as of 14:37, 27 July 2008
Template:STRUCTURE 2d0k
Methionine-free mutant of Escherichia coli dihydrofolate reductase
Template:ABSTRACT PUBMED 16510443
About this Structure
2D0K is a Single protein structure of sequence from Escherichia coli. Full crystallographic information is available from OCA.
Reference
Evolutional design of a hyperactive cysteine- and methionine-free mutant of Escherichia coli dihydrofolate reductase., Iwakura M, Maki K, Takahashi H, Takenawa T, Yokota A, Katayanagi K, Kamiyama T, Gekko K, J Biol Chem. 2006 May 12;281(19):13234-46. Epub 2006 Mar 1. PMID:16510443
Page seeded by OCA on Sun Jul 27 17:37:32 2008
