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M. L. di Salvo - Synthesis of pyridoxal phosphate in the vitamin B 6 salvage pathway
use it as cofactor in the form of a complex with the
specific substrates. We have recently carried out
experiments with both human and E. coli PNPOx and
shown that the human enzyme is more efficient in
transferring PLP to human PLP-dependent enzymes,
whereas E. coli PNPOx transfers PLP better to bacterial
enzymes. This results strongly suggest an
enzyme-enzyme recognition step, in which PLP may
be transferred directly to the apo-enzymes through a
channelling mechanism. The encounter between PLP
and apo-proteins in the cell may take place at different
stages of the polypeptide folding process and
affect it to a variable extent. Apparently, the role of
PLP in folding and structural stabilization varies
among the enzymes which use it as cofactor. This is
not surprising, considering that there are at least five
evolutionary unrelated families of such enzymes, corresponding
to completely different protein folds. Our
research group has focused attention on serine
hydroxymethyltransferase (SHMT), which may be
taken as a model for the whole fold type I family. The
folding mechanism of E. coli SHMT has been investigated
in detail and is understood better than that of
any other fold-type I enzyme. Our studies on the
mechanism of addition of PLP to folding apo-SHMT
involve several aspects: the role of conserved
hydrophobic clusters in the late phase of folding and
PLP binding; the structural requisites involved in the
recognition process between PLP and apo-SHMT;
the detailed studies on the structural changes taking
place in folding and PLP binding; and the characterisation
of human S394N and L474F SHMT mutants
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with respect to the kinetics of PLP addition. These
two single nucleotide polymorphisms of the gene
encoding cytosolic SHMT, may be associated with
several disease states, such as neural tube defects, cardio
vascular diseases and pregnancy complications,
connected to hyperhomocysteinemia.
Our research project also involves human PLK, the
other enzyme involved in the vitamin B 6 salvage
pathway. Recently, a direct inhibitory effect of several
drugs on PLK has been shown, which results in
vitamin B 6 deficiency and various side effects related
to the central nervous system. Well known examples
are theophylline, used to treat asthma, and progabide
to treat epilepsy. A number of polymorphisms were
also discovered in the PLK coding gene. Our work
has focused on the delucidation of the mechanism of
action of this enzyme, with particular interest on the
residue D235 which was shown to play a major role
in the deprotonation step of the substrate hydroxyl
group prior to nucleophilic attack the γ-phosphate
group of ATP. We are currently carrying out structural
and functional requirement studies on the
interaction between PLK and the several drugs that
modutale its activity.
Selected publications
Gandhi AK, Ghatge M, Musayev FN, Sease A,
Aboagye SO, di Salvo ML, Schirch V, Safo MK.
Kinetic and Structural Studies of the Role of the
Active Site Residue Asp235 of Human Pyridoxal
Kinase. Biochem Biophys Res Commun., in press.