download report - Istituto Pasteur

P a r t i c i p a n t s :
Francesco Bossa, professor; Roberto Contestabile,
Sebastiana Angelaccio, researchers; Rita Florio, Mirella
Vivoli, PhD students.
C o l l a b o r a t i o n s :
Department of Medicinal Chemistry, Virginia Commonwealth
University, USA (Prof. Martin Safo); Department of Medical
Laboratory Science and Biotechnology, National Cheng Kung
University, Taiwan (Prof. Tzu-Fan Fu).
Report of activity
Pyridoxal 5'-phosphate (PLP) was first identified in
the mid forties as the cofactor required for the biochemical
reaction of transamination. Since then,
PLP-dependent enzymes have been the subject of
extensive research. The interest aroused by these
enzymes comes from their unequalled catalytic versatility
and widespread involvement in cellular
processes. Nowadays, more than 140 different
enzyme activities based on PLP are classified by the
Enzyme Commission and represent 4% of all known
catalytic activities. PLP-dependent enzymes are not
only involved in the synthesis, interconversion and
degradation of amino acids (among which neurotransmitters)
but also play key roles in the metabolism
of one-carbon units, biogenic amines,
tetrapyrrolic compounds, amino-sugars, modulation
of steroid receptor-mediated gene expression and
regulation of immune function. While bacteria,
plants and fungi synthesize PLP, animals obtain this
cofactor from B 6 vitamers acquired from the diet
and recycled in a salvage pathway, in which two
enzymes are essentially involved: pyridoxal kinase
(PLK) and pyridoxamine phosphate oxidase
(PNPOx). Once made available, PLP is somehow
targeted to the dozens different apo-B 6 enzymes
that are being synthesized in the cell. Crucial and
poorly answered questions are how PLP availability
83
Molecular recognition in biomolecules - AREA 4
Synthesis of pyridoxal phosphate in the vitamin B 6 salvage
pathway and targeting of the cofactor to apo-enzymes
Principal investigator: Martino Luigi di Salvo
Researcher in Biochemistry
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”
Tel: (+39) 06 49917684; Fax: (+39) 06 49917566
martino.disalvo@uniroma1.it
is regulated and how the cofactor reacts with the
apo-enzymes while these are folding. Dietary deficiency
of vitamin B 6 or inborn defects in the
enzymes of the salvage pathway result in severe
neurological dysfunctions. The study of inborn
errors affecting PLP availability may provide a
unique opportunity for studying the role of PLP as
a regulator of enzyme activities in man. Autosomal
recessive mutations in the gene encoding PNPOx
cause neonatal epileptic encephalopathy (NEE).
Individuals affected by NEE were found to be
homozygote for missense, splice site and stop codon
mutations. One of these mutations is R229, the last
amino acid in a short sequence which is completely
conserved in all known species. Structural studies on
recombinant hPNPOx have shown that R229 might
be involved in the tight binding of cofactor FMN to
the active site of the enzyme, by direct interaction
with the phosphate group of FMN. Substitution of
Arg with Trp is predicted to disrupt this interaction
and that of other amino acids in the vicinity. We
have expressed, purified, functionally and structurally
characterized human PNPOx R229W and
R229Q mutants. Dissociation constants, kinetic and
crystal structure analyses show that the enzyme is
still able to bind the cofactor, but its catalytic activity
is greately impaired mostly because of its low
capability of binding the substrate.
Another important aspect of B 6 related metabolic
dysfunctions is that some patients with inborn errors
affecting PLP-dependent enzymes are responsive to
pyridoxine treatment, suggesting that the mutations
may impair the binding of the cofactor. Moreover,
PLP may play a role in assisting protein folding and
stability. Although much work has been done on the
mechanism and structure of B 6 enzymes, little is
known on how the apo-forms are converted to the
holo-forms while they are folding. According to some
authors, PLP may be transferred by PNPOx directly
to the apo-enzymes which require it as cofactor.
Alternatively, PLP may react with the enzymes that