6 research outputs found
New Functional Aspects of the Atypical Protein Tyrosine Phosphatase VHZ
LDP3
(VHZ) is the smallest classical protein tyrosine phosphatase
(PTP) known to date and was originally misclassified as an atypical
dual-specificity phosphatase. Kinetic isotope effects with steady-state
and pre-steady-state kinetics of VHZ and mutants with <i>p</i>-nitrophenol phosphate have revealed several unusual properties.
VHZ is significantly more active than previously reported but remains
one of the least active PTPs. Highly unusual for a PTP, VHZ possesses
two acidic residues (E134 and D65) in the active site. D65 occupies
the position corresponding to the typical general acid in the PTP
family. However, VHZ primarily utilizes E134 as the general acid,
with D65 taking over this role when E134 is mutated. This unusual
behavior is facilitated by two coexisting, but unequally populated,
substrate binding modes. Unlike most classical PTPs, VHZ exhibits
phosphotransferase activity. Despite the presence of the Q-loop that
normally prevents alcoholysis of the phosphoenzyme intermediate in
other classical PTPs, VHZ readily phosphorylates ethylene glycol.
Although mutations of Q-loop residues affect this phosphotransferase
activity, mutations on the IPD loop that contains the general acid
exert more control over this process. A single P68V substitution on
this loop completely abolishes phosphotransferase activity. The ability
of native VHZ to catalyze transphosphorylation may lead to an imbalance
of intracellular phosphorylation, which could explain the correlation
of its overexpression with several types of cancer
New Aspects of the Phosphatase VHZ Revealed by a High-Resolution Structure with Vanadate and Substrate Screening
The recently discovered 150-residue human VHZ (VH1-related
protein,
Z member) is one of the smallest protein tyrosine phosphatases (PTPs)
known and contains only the minimal structural elements common to
all PTPs. We report a substrate screening analysis and a crystal structure
of the VHZ complex with vanadate at 1.1 Ă
resolution, with a
detailed structural comparison with other members of the protein tyrosine
phosphatase family, including classical tyrosine-specific protein
tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs).
A screen with 360 phosphorylated peptides shows VHZ efficiently catalyzes
the hydrolysis of phosphotyrosine (pY)-containing peptides but exhibits
no activity toward phosphoserine (pS) or phosphothreonine (pT) peptides.
The new structure reveals a deep and narrow active site more typical
of the classical tyrosine-specific PTPs. Despite the high degrees
of structural and sequence similarity between VHZ and classical PTPs,
its general acid IPD-loop is most likely conformationally rigid, in
contrast to the flexible WPD counterpart of classical PTPs. VHZ also
lacks substrate recognition domains and other domains typically found
on classical PTPs. It is therefore proposed that VHZ is more properly
classified as an atypical PTP rather than an atypical DSP, as has
been suggested
Metavanadate at the Active Site of the Phosphatase VHZ
Vanadate is a potent modulator of a number of biological
processes
and has been shown by crystal structures and NMR spectroscopy to interact
with numerous enzymes. Although these effects often occur under conditions
where oligomeric forms dominate, the crystal structures and NMR data
suggest that the inhibitory form is usually monomeric orthovanadate,
a particularly good inhibitor of phosphatases because of its ability
to form stable trigonal-bipyramidal complexes. We performed a computational
analysis of a 1.14 Ă
structure of the phosphatase VHZ in complex
with an unusual metavanadate species and compared it with two classical
trigonal-bipyramidal vanadateâphosphatase complexes. The results
support extensive delocalized bonding to the apical ligands in the
classical structures. In contrast, in the VHZ metavanadate complex,
the central, planar VO<sub>3</sub><sup>â</sup> moiety has only
one apical ligand, the nucleophilic Cys95, and a gap in electron density
between V and S. A computational analysis showed that the VâS
interaction is primarily ionic. A mechanism is proposed to explain
the formation of metavanadate in the active site from a dimeric vanadate
species that previous crystallographic evidence has shown to be able
to bind to the active sites of phosphatases related to VHZ. Together,
the results show that the interaction of vanadate with biological
systems is not solely reliant upon the prior formation of a particular
inhibitory form in solution. The catalytic properties of an enzyme
may act upon the oligomeric forms primarily present in solution to
generate species such as the metavanadate ion observed in the VHZ
structure
Conservative Tryptophan Mutants of the Protein Tyrosine Phosphatase YopH Exhibit Impaired WPD-Loop Function and Crystallize with Divanadate Esters in Their Active Sites
Catalysis in protein tyrosine phosphatases
(PTPs) involves movement
of a protein loop called the WPD loop that brings a conserved aspartic
acid into the active site to function as a general acid. Mutation
of the tryptophan in the WPD loop of the PTP YopH to any other residue
with a planar, aromatic side chain (phenylalanine, tyrosine, or histidine)
disables general acid catalysis. Crystal structures reveal these conservative
mutations leave this critical loop in a catalytically unproductive,
quasi-open position. Although the loop positions in crystal structures
are similar for all three conservative mutants, the reasons inhibiting
normal loop closure differ for each mutant. In the W354F and W354Y
mutants, steric clashes result from six-membered rings occupying the
position of the five-membered ring of the native indole side chain.
The histidine mutant dysfunction results from new hydrogen bonds stabilizing
the unproductive position. The results demonstrate how even modest
modifications can disrupt catalytically important protein dynamics.
Crystallization of all the catalytically compromised mutants in the
presence of vanadate gave rise to vanadate dimers at the active site.
In W354Y and W354H, a divanadate ester with glycerol is observed.
Such species have precedence in solution and are known from the small
molecule crystal database. Such species have not been observed in
the active site of a phosphatase, as a functional phosphatase would
rapidly catalyze their decomposition. The compromised functionality
of the mutants allows the trapping of species that undoubtedly form
in solution and are capable of binding at the active sites of PTPs,
and, presumably, other phosphatases. In addition to monomeric vanadate,
such higher-order vanadium-based molecules are likely involved in
the interaction of vanadate with PTPs in solution
Mechanistic Aspects of Phosphate Diester Cleavage Assisted by Imidazole. A Template Reaction for Obtaining Aryl Phosphoimidazoles
Phosphoimidazole-containing compounds
are versatile players in
biological and chemical processes. We explore catalytic and mechanistic
criteria for the efficient formation of cyclic aryl phosphoimidazoles
in aqueous solution, viewed as a template reaction for the in situ
synthesis of related compounds. To provide a detailed analysis for
this reaction a series of <i>o</i>-(2â˛-imidazolyl)Ânaphthyl
(4-nitrophenyl) phosphate isomers were examined to provide a basis
for analysis of both mechanism and the influence of structural factors
affecting the nucleophilic attack of the imidazolyl group on the phosphorus
center of the substrate. Formation of the cyclic aryl phosphoimidazoles
was probed by NMR and ESI-MS techniques. Kinetic experiments show
that cyclization is faster under alkaline conditions, with an effective
molarity up to 2900 M for the imidazolyl group, ruling out competition
from external nucleophiles. Heavy atom isotope effect and computational
studies show that the reaction occurs through a S<sub>N</sub>2Â(P)-type
mechanism involving a pentacoordinated phosphorus TS, with apical
positions occupied by the incoming imidazolyl nucleophile and the <i>p</i>-nitrophenolate leaving group. The PâO bond to the
leaving group is about 50â60% broken in the transition state
Structural and Kinetic Properties of the Aldehyde Dehydrogenase NahF, a Broad Substrate Specificity Enzyme for Aldehyde Oxidation
The
salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation
of salicylaldehyde to salicylate using NAD<sup>+</sup> as a cofactor,
the last reaction of the upper degradation pathway of naphthalene
in <i>Pseudomonas putida</i> G7. The naphthalene is an abundant
and toxic compound in oil and has been used as a model for bioremediation
studies. The steady-state kinetic parameters for oxidation of aliphatic
or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF
catalyzes the oxidation of aromatic aldehydes with large <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> values close to 10<sup>6</sup> M<sup>â1</sup> s<sup>â1</sup>. The active site
of NahF is highly hydrophobic, and the enzyme shows higher specificity
for less polar substrates than for polar substrates, e.g., acetaldehyde.
The enzyme shows ι/β folding with three well-defined domains:
the oligomerization domain, which is responsible for the interlacement
between the two monomers; the Rossmann-like fold domain, essential
for nucleotide binding; and the catalytic domain. A salicylaldehyde
molecule was observed in a deep pocket in the crystal structure of
NahF where the catalytic C284 and E250 are present. Moreover, the
residues G150, R157, W96, F99, F274, F279, and Y446 were thought to
be important for catalysis and specificity for aromatic aldehydes.
Understanding the molecular features responsible for NahF activity
allows for comparisons with other aldehyde dehydrogenases and, together
with structural information, provides the information needed for future
mutational studies aimed to enhance its stability and specificity
and further its use in biotechnological processes