PDBsum entry 1h4d

Molybdenum cofactor biosynthesis

PDB id: 1h4d

Contents

Protein chain

188 a.a. *

Ligands

CIT ×2

Metals

_LI

Waters ×185

* Residue conservation analysis

PDB id:

1h4d

Name:

Molybdenum cofactor biosynthesis

Title:

Biochemical and structural analysis of the molybdenum cofactor biosynthesis protein moba

Structure:

Molybdopterin-guanine dinucleotide biosynthesis protein a. Chain: a. Synonym: protein fa. Engineered: yes. Mutation: yes. Other_details: the full length construct was c-terminally extended with a 7-residue nickel affinity tag of sequence ser-his-his-his-his- his-his.

Source:

Escherichia coli. Organism_taxid: 562. Strain: jm109. Variant: m15[prep4]. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_variant: m15[prep4]. Other_details: mutation introduced by 3-way pcr and verified by DNA sequencing

Resolution:

1.74Å R-factor: 0.175 R-free: 0.219

Authors:

A.Guse,C.E.M.Stevenson,J.Kuper,G.Buchanan,G.Schwarz,R.R.Mendel, D.M.Lawson,T.Palmer

Key ref:

A.Guse et al. (2003). Biochemical and structural analysis of the molybdenum cofactor biosynthesis protein MobA. J Biol Chem, 278, 25302-25307. PubMed id: 12719427 DOI: 10.1074/jbc.M302639200

Date:

26-Feb-03 Release date: 08-May-03

Protein chain

P32173  (MOBA_ECOLI) -  Molybdenum cofactor guanylyltransferase from Escherichia coli (strain K12)

Seq: 194 a.a.

Struc: 188 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.2.7.7.77 - molybdenum cofactor guanylyltransferase.
• Reaction: Mo-molybdopterin + GTP + H⁺ = Mo-molybdopterin guanine dinucleotide + diphosphate

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

Added reference

DOI no: 10.1074/jbc.M302639200

J Biol Chem 278:25302-25307 (2003)

PubMed id: 12719427

Biochemical and structural analysis of the molybdenum cofactor biosynthesis protein MobA.

A.Guse, C.E.Stevenson, J.Kuper, G.Buchanan, G.Schwarz, G.Giordano, A.Magalon, R.R.Mendel, D.M.Lawson, T.Palmer.

ABSTRACT

Molybdopterin guanine dinucleotide (MGD) is the form of the molybdenum cofactor that is required for the activity of most bacterial molybdoenzymes. MGD is synthesized from molybdopterin (MPT) and GTP in a reaction catalyzed by the MobA protein. Here we report that wild type MobA can be copurified along with bound MPT and MGD, demonstrating a tight binding of both its substrate and product. To study structure-function relationships, we have constructed a number of site-specific mutations of the most highly conserved amino acid residues of the MobA protein family. Variant MobA proteins were characterized for their ability to support the synthesis of active molybdenum enzymes, to bind MPT and MGD, to interact with the molybdenum cofactor biosynthesis proteins MobB and MoeA. They were also characterized by x-ray structural analysis. Our results suggest an essential role for glycine 15 of MobA, either for GTP binding and/or catalysis, and an involvement of glycine 82 in the stabilization of the product-bound form of the enzyme. Surprisingly, the individual and double substitution of asparagines 180 and 182 to aspartate did not affect MPT binding, catalysis, and product stabilization.

Selected figure(s)

Figure 1.
FIG. 1. Alignment of MobA amino acid sequences from bacteria. The abbreviations used are: Eco, E. coli (17); Rsp, Rhodobacter sphaeroides (41); Ppu, Pseudomonas putida (GenBankTM/EBI accession number AJ2429522); Bsu, Bacillus subtilis (GenBankTM/EBI accession number AAC24900 [GenBank] ). Conserved residues that were subjected to mutagenesis in this study are indicated by asterisks under the sequence.

Figure 5.
FIG. 5. Structural analysis of amino acid substituted MobA proteins. Composite figure showing the locations of the substituted residues in MobA and their effects on the local structure. The central picture shows a C trace of the wild type structure in white (Protein Data Bank accession code 1E5K [PDB] ), with the side chains (or C s for glycines) of the substituted residues shown in black. The four circular insets show the local changes in the structures for four of the five variants whose structures were determined: close-ups of the wild type (white) and variant (black) structures are superposed. The R19A variant is not shown, since the resultant model was virtually indistinguishable from the wild type. In fact, the side chain of Arg 19 was not visible in the electron density maps for any of the crystal structures of MobA (including wild type), although it is included in the central picture for completeness. Note the change in the conformation of the consensus loop in the G22L variant and the loss of a salt bridge in the D101N form of the protein. The perturbations in the N180D and N182D structures are relatively minor. The figure was generated using SwissPDBviewer (40) (www.expasy.ch/spdbv) and rendered using POV-RayTM (www.povray.org).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2003, 278, 25302-25307) copyright 2003.

Figures were selected by an automated process.