 |
PDBsum entry 1h4d
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Molybdenum cofactor biosynthesis
|
PDB id
|
|
|
|
1h4d
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
Contents |
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
* Residue conservation analysis
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
Biochemical and structural analysis of the molybdenum cofactor biosynthesis protein moba.
|
|
|
Authors
|
|
A.Guse,
C.E.Stevenson,
J.Kuper,
G.Buchanan,
G.Schwarz,
G.Giordano,
A.Magalon,
R.R.Mendel,
D.M.Lawson,
T.Palmer.
|
|
|
Ref.
|
|
J Biol Chem, 2003,
278,
25302-25307.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
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.
|
|
|
|
|
|
|
|
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.
|
|
|
|
|
|
|
