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Tyr 64, hydrogen-bonded to coenzyme phosphate in Treponema denticola cystalysin is changed to alanine by site-directed mutagenesis, spectroscopic and kinetic properties of the Tyr 64 are investigated to explore the differences in coenzyme structure and kinetic mechanism to those of wild type enzyme. The result finds new insight into the catalytic mechanism of cystalysin and highlights the functional role of Tyr 64.

Research has been conducted on Treponema denticola cystalysin. The functional properties of this cystalysin have been investigated via the pH- and ligand-induced spectral transition analysis and the results suggest that cystalysin is more catalytically competent in alkaline solution.

The inactivation of dopa decarboxylase (DDC) by the inhibitors serotonin and dopamine was investigated. Recombinant DDC was purified to homogeneity with slight modification for use in the study. Results showed that the kinetics of DDC inactivation by serotonin and dopamine is consistent with the conversion of both inhibitors to 5-hydroxyindolacetaldehyde and 3,4-dihydroxyphenylacetaldehyde, respectively. In addition, the mechanism of DDC inactivation probably involves generation of an active site directed affinity label, 3,4-dihydroxyphenylacetone.

The unusual oxygen-consuming oxidative deamination reaction catalyzed by the pyridoxal 5'-phosphate (PLP) enzyme DOPA decarboxylase (DDC) is examined. The catalytic properties of the T246A DDC mutant has shown that the Thr246-His192 dyad could act as a general base in promoting the first step of the oxidative deamination of aromatic amines.

The protein misfolding cyclic amplification (PMCA) and scrapie cell assay (SCA) techniques are used to study the role of N-linked glycosylation on prion formation in vitro. The results have shown that the interactions between different Pr[P.sup.C] (host-encoded glycoprotein) glycoforms control the efficiency of prion formation in a species-specific manner.

Pyridoxal 5′-phosphate (PLP)-dependent enzymes catalyze a wide range of reactions of amino acids and amines, with the exception of glycogen phosphorylase which exhibits peculiar both substrate preference and chemical mechanism. They represent about 4% of the gene products in eukaryotic cells. Although structure-function investigations regarding these enzymes are copious, their regulation by post-translational modifications is largely unknown. Protein phosphorylation is the most common post-translational modification fundamental in mediating diverse cellular functions. This review aims at summarizing the current knowledge on regulation of PLP enzymes by phosphorylation. Starting from the paradigmatic PLP-dependent glycogen phosphorylase, the first phosphoprotein discovered, we collect data in literature regarding functional phosphorylation events of eleven PLP enzymes belonging to different fold types and discuss the impact of the modification in affecting their activity and localization as well as the implications on the pathogenesis of diseases in which many of these enzymes are involved. The pivotal question is to correlate the structural consequences of phosphorylation among PLP enzymes of different folds with the functional modifications exerted in terms of activity or conformational changes or others. Although the literature shows that the phosphorylation of PLP enzymes plays important roles in mediating diverse cellular functions, our recapitulation of clue findings in the field makes clear that there is still much to be learnt. Besides mass spectrometry-based proteomic analyses, further biochemical and structural studies on purified native proteins are imperative to fully understand and predict how phosphorylation regulates PLP enzymes and to find the relationship between addition of a phosphate moiety and physiological response. [ABSTRACT FROM AUTHOR]

Dopa decarboxylase (DDC), a pyridoxal 5'-phosphate (PLP) enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinson's disease (PD). PD is a neurodegenerative disease mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide) is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide) are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a) to use virtual screening to identify potential human DDC inhibitors and (b) to evaluate the reliability of our virtualscreening (VS) protocol by experimentally testing the ''in vitro'' activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with Ki values ⩾10 μM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with Ki values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a Ki value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery. [ABSTRACT FROM AUTHOR]

G41 is an interfacial residue located within the α-helix 34-42 of alanine:glyoxylate aminotransferase (AGT). Its mutations on the major (AGT-Ma) or the minor (AGT-Mi) allele give rise to the variants G41R-Ma, G41R-Mi, and G41V-Ma causing hyperoxaluria type 1. Impairment of dimerization in these variants has been suggested to be responsible for immunoreactivity deficiency, intraperoxisomal aggregation, and sensitivity to proteasomal degradation. However, no experimental evidence supports this view. Here we report that G41 mutations, besides increasing the dimer-monomer equilibrium dissociation constant, affect the protein conformation and stability, and perturb its active site. As compared to AGT-Ma or AGT-Mi, G41 variants display different near-UV CD and intrinsic emission fluorescence spectra, larger exposure of hydrophobic surfaces, sensitivity to Met53-Tyr54 peptide bond cleavage by proteinase K, decreased thermostability, reduced coenzyme binding affinity, and catalytic efficiency. Additionally, unlike AGT-Ma and AGT-Mi, G41 variants under physiological conditions form insoluble inactive high-order aggregates (∼5, 000 nm) through intermolecular electrostatic interactions. A comparative molecular dynamics study of the putative structures of AGT-Mi and G41R-Mi predicts that G41 → R mutation causes a partial unwinding of the 34-42 a-helix and a displacement of the first 44 N-terminal residues including the active site loop 24-32. These simulations help us to envisage the possible structural basis of AGT dysfunction associated with G41 mutations. The detailed insight into how G41 mutations act on the structure-function of AGT may contribute to achieve the ultimate goal of correcting the effects of these mutations. [ABSTRACT FROM AUTHOR]