1m8k
From Proteopedia
(New page: 200px<br /><applet load="1m8k" size="450" color="white" frame="true" align="right" spinBox="true" caption="1m8k, resolution 3.00Å" /> '''Crystal Structure Of...) |
|||
| Line 1: | Line 1: | ||
| - | [[Image:1m8k.gif|left|200px]]<br /><applet load="1m8k" size=" | + | [[Image:1m8k.gif|left|200px]]<br /><applet load="1m8k" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1m8k, resolution 3.00Å" /> | caption="1m8k, resolution 3.00Å" /> | ||
'''Crystal Structure Of Methanobacterium Thermoautotrophicum Nicotinamide Mononucleotide Adenylyltransferase Mutant H19A complexed with NAD'''<br /> | '''Crystal Structure Of Methanobacterium Thermoautotrophicum Nicotinamide Mononucleotide Adenylyltransferase Mutant H19A complexed with NAD'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Several residues lining the ATP-binding site of Methanobacterium | + | Several residues lining the ATP-binding site of Methanobacterium thermoautotrophicum nicotinamide mononucleotide adenylyltransferase (NMNATase) were mutated in an effort to better characterize their roles in substrate binding and catalysis. Residues selected were Arg-11 and Arg-136, both of which had previously been implicated as substrate binding residues, as well as His-16 and His-19, part of the HXGH active site motif and postulated to be of importance in catalysis. Kinetic studies revealed that both Arg-11 and Arg-136 contributed to the binding of the substrate, ATP. When these amino acids were replaced by lysines, the apparent Km values of the respective mutants for ATP decreased by factors of 1.3 and 2.9 and by factors of 1.9 and 8.8 when the same residues were changed to alanines. All four Arg mutants displayed unaltered Km values for NMN. The apparent kcat values of the R11K and R136K mutants were the same as those of WT NMNATase but the apparent kcat values of the alanine mutants had decreased. Crystal structures of the Arg mutants revealed NAD+ and SO42- molecules trapped at their active sites. The binding interactions of NAD+ were unchanged but the binding of SO42- was altered in these mutants compared with wild type. The alanine mutants at positions His-16 and His-19 retained approximately 6 and 1.3%, respectively, of WT NMNATase activity indicating that His-19 is a key catalytic group. Surprisingly, this H19A mutant displayed a novel and distinct mode of NAD+ binding when co-crystallized in the presence of NAD+ and SO42-. |
==About this Structure== | ==About this Structure== | ||
| - | 1M8K is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Methanothermobacter_thermautotrophicus Methanothermobacter thermautotrophicus] with SO4 and NAD as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Nicotinamide-nucleotide_adenylyltransferase Nicotinamide-nucleotide adenylyltransferase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.7.1 2.7.7.1] Full crystallographic information is available from [http:// | + | 1M8K is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Methanothermobacter_thermautotrophicus Methanothermobacter thermautotrophicus] with <scene name='pdbligand=SO4:'>SO4</scene> and <scene name='pdbligand=NAD:'>NAD</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Nicotinamide-nucleotide_adenylyltransferase Nicotinamide-nucleotide adenylyltransferase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.7.1 2.7.7.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1M8K OCA]. |
==Reference== | ==Reference== | ||
| Line 14: | Line 14: | ||
[[Category: Nicotinamide-nucleotide adenylyltransferase]] | [[Category: Nicotinamide-nucleotide adenylyltransferase]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Pai, E | + | [[Category: Pai, E F.]] |
[[Category: Saridakis, V.]] | [[Category: Saridakis, V.]] | ||
[[Category: NAD]] | [[Category: NAD]] | ||
| Line 20: | Line 20: | ||
[[Category: nucleotidyltransferase hxgh active site motif]] | [[Category: nucleotidyltransferase hxgh active site motif]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:52:44 2008'' |
Revision as of 11:52, 21 February 2008
|
Crystal Structure Of Methanobacterium Thermoautotrophicum Nicotinamide Mononucleotide Adenylyltransferase Mutant H19A complexed with NAD
Overview
Several residues lining the ATP-binding site of Methanobacterium thermoautotrophicum nicotinamide mononucleotide adenylyltransferase (NMNATase) were mutated in an effort to better characterize their roles in substrate binding and catalysis. Residues selected were Arg-11 and Arg-136, both of which had previously been implicated as substrate binding residues, as well as His-16 and His-19, part of the HXGH active site motif and postulated to be of importance in catalysis. Kinetic studies revealed that both Arg-11 and Arg-136 contributed to the binding of the substrate, ATP. When these amino acids were replaced by lysines, the apparent Km values of the respective mutants for ATP decreased by factors of 1.3 and 2.9 and by factors of 1.9 and 8.8 when the same residues were changed to alanines. All four Arg mutants displayed unaltered Km values for NMN. The apparent kcat values of the R11K and R136K mutants were the same as those of WT NMNATase but the apparent kcat values of the alanine mutants had decreased. Crystal structures of the Arg mutants revealed NAD+ and SO42- molecules trapped at their active sites. The binding interactions of NAD+ were unchanged but the binding of SO42- was altered in these mutants compared with wild type. The alanine mutants at positions His-16 and His-19 retained approximately 6 and 1.3%, respectively, of WT NMNATase activity indicating that His-19 is a key catalytic group. Surprisingly, this H19A mutant displayed a novel and distinct mode of NAD+ binding when co-crystallized in the presence of NAD+ and SO42-.
About this Structure
1M8K is a Single protein structure of sequence from Methanothermobacter thermautotrophicus with and as ligands. Active as Nicotinamide-nucleotide adenylyltransferase, with EC number 2.7.7.1 Full crystallographic information is available from OCA.
Reference
Mutational, structural, and kinetic studies of the ATP-binding site of Methanobacterium thermoautotrophicum nicotinamide mononucleotide adenylyltransferase., Saridakis V, Pai EF, J Biol Chem. 2003 Sep 5;278(36):34356-63. Epub 2003 Jun 16. PMID:12810729
Page seeded by OCA on Thu Feb 21 13:52:44 2008
