PDBsum entry 1m8f

Transferase

PDB id: 1m8f

Contents

Protein chain

167 a.a. *

Ligands

NAD

Waters ×98

* Residue conservation analysis

PDB id:

1m8f

Name:

Transferase

Title:

Crystal structure of methanobacterium thermoautotrophicum nicotinamide mononucleotide adenylyltransferase mutant r11a complexed with NAD

Structure:

Nicotinamide-nucleotide adenylyltransferase. Chain: a. Synonym: NAD(+) pyrophosphorylase, NAD(+) diphosphorylase, nmn adenylyltransferase. Engineered: yes. Mutation: yes

Source:

Methanothermobacter thermautotrophicus. Organism_taxid: 145262. Gene: mth150. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Biol. unit:

Hexamer (from PDB file)

Resolution:

2.40Å R-factor: 0.194 R-free: 0.246

Authors:

V.Saridakis,E.F.Pai

Key ref:

V.Saridakis and E.F.Pai (2003). Mutational, structural, and kinetic studies of the ATP-binding site of Methanobacterium thermoautotrophicum nicotinamide mononucleotide adenylyltransferase. J Biol Chem, 278, 34356-34363. PubMed id: 12810729 DOI: 10.1074/jbc.M205369200

Date:

24-Jul-02 Release date: 15-Jul-03

Protein chain

O26253  (NADM_METTH) -  Nicotinamide-nucleotide adenylyltransferase from Methanothermobacter thermautotrophicus (strain ATCC 29096 / DSM 1053 / JCM 10044 / NBRC 100330 / Delta H)

Seq: 178 a.a.

Struc: 167 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.2.7.7.1 - nicotinamide-nucleotide adenylyltransferase.
• Reaction: beta-nicotinamide D-ribonucleotide + ATP + H⁺ = diphosphate + NAD⁺

beta-nicotinamide D-ribonucleotide + ATP + H(+) = diphosphate + NAD(+) (Bound ligand (Het Group name = NAD) corresponds exactly)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.M205369200

J Biol Chem 278:34356-34363 (2003)

PubMed id: 12810729

Mutational, structural, and kinetic studies of the ATP-binding site of Methanobacterium thermoautotrophicum nicotinamide mononucleotide adenylyltransferase.

V.Saridakis, E.F.Pai.

ABSTRACT

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-.

Selected figure(s)

Figure 2.
FIG. 2. A, the crystallographic contacts formed between adjacent WT hexamers complexed with NAD^+ are shown (yellow and red). B, the principle interactions between the hexamers are shown in greater detail with the residues forming these interactions in ball and stick form.

Figure 3.
FIG. 3. A, the crystallographic contacts formed between adjacent H19A hexamers complexed with NAD^+ are shown (blue and green). B, the principle interactions between the hexamers are shown in greater detail with the residues forming these interactions in ball and stick form.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2003, 278, 34356-34363) copyright 2003.

Figures were selected by an automated process.