A comprehensive picture of the mutations associated with aromatic amino acid decarboxylase deficiency: from molecular mechanisms to therapy implications.

Dopa decarboxylase (DDC), or aromatic amino acid decarboxylase (AADC), is a pyridoxal 5'-phosphate enzyme responsible for the production of the neurotransmitters dopamine and serotonin. Deficit of this enzyme causes AADC deficiency, an inherited neurometabolic disorder. To date, 18 missense homozygous mutations have been identified through genetic screening in ∼80 patients. However, little is known about the mechanism(s) by which mutations cause disease. Here we investigated the impact of these pathogenic mutations and of an artificial one on the conformation and the activity of wild-type DDC by a combined approach of bioinformatic, spectroscopic and kinetic analyses. All mutations reduce the kcat value, and, except the mutation R347Q, alter the tertiary structure, as revealed by an increased hydrophobic surface and a decreased near-UV circular dichroism signal. The integrated analysis of the structural and functional consequences of each mutation strongly suggests that the reason underlying the pathogenicity of the majority of disease-causing mutations is the incorrect apo-holo conversion. In fact, the most remarkable effects are seen upon mutation of residues His70, His72, Tyr79, Phe80, Pro81, Arg462 and Arg447 mapping to or directly interacting with loop1, a structural key element involved in the apo-holo switch. Instead, different mechanisms are responsible for the pathogenicity of R347Q, a mere catalytic mutation, and of L38P and A110Q mutations causing structural-functional defects. These are due to local perturbation transmitted to the active site, as predicted by molecular dynamic analyses. Overall, the results not only give comprehensive molecular insights into AADC deficiency, but also provide an experimental framework to suggest appropriate therapeutic treatments.

Role of active-site residues Tyr55 and Tyr114 in catalysis and substrate specificity of Corynebacterium diphtheriae C-S lyase.

In recent years, there has been increased interest in bacterial methionine biosynthesis enzymes as antimicrobial targets because of their pivotal role in cell metabolism. C-S lyase from Corynebacterium diphtheriae is a pyridoxal 5'-phosphate-dependent enzyme in the transsulfuration pathway that catalyzes the α,ß-elimination of sulfur-containing amino acids, such as L-cystathionine, to generate ammonia, pyruvate, and homocysteine, the immediate precursor of L-methionine. In order to gain deeper insight into the functional and dynamic properties of the enzyme, mutants of two highly conserved active-site residues, Y55F and Y114F, were characterized by UV-visible absorbance, fluorescence, and CD spectroscopy in the absence and presence of substrates and substrate analogs, as well as by steady-state kinetic studies. Substitution of Tyr55 with Phe apparently causes a 130-fold decrease in K(d)(PLP) at pH 8.5 providing evidence that Tyr55 plays a role in cofactor binding. Moreover, spectral data show that the mutant accumulates the external aldimine intermediate suggesting that the absence of interaction between the hydroxyl moiety and PLP-binding residue Lys222 causes a decrease in the rate of substrate deprotonation. Mutation of Tyr114 with Phe slightly influences hydrolysis of L-cystathionine, and causes a change in substrate specificity towards L-serine and O-acetyl-L-serine compared to the wild type enzyme. These findings, together with computational data, provide useful insights in the substrate specificity of C-S lyase, which seems to be regulated by active-site architecture and by the specific conformation in which substrates are bound, and will aid in development of inhibitors.

Identification of aldolase and ferredoxin reductase within the dbt operon of Burkholderia fungorum DBT1.

Burkholderia fungorum DBT1, first isolated from settling particulate matter of an oil refinery wastewater, is a bacterial strain which has been shown capable of utilizing several polycyclic aromatic hydrocarbons (PAHs) including dibenzothiophene (DBT). In particular, this microbe is able to efficiently degrade DBT through the Kodama pathway. Previous investigations have lead to the identification of six genes, on a total of eight, required for DBT degradation. In the present study, a combined experimental/computational approach was adopted to identify and in silico characterize the two missing genes, namely a ferredoxin reductase and a hydratase-aldolase. Thus, the finding of all enzymatic components of the Kodama pathway in B. fungorum DBT1 makes this bacterial strain amenable for possible exploitation in soil bioremediation protocols.

Genome-wide Membrane Protein Structure Prediction.

Transmembrane proteins allow cells to extensively communicate with the external world in a very accurate and specific way. They form principal nodes in several signaling pathways and attract large interest in therapeutic intervention, as the majority pharmaceutical compounds target membrane proteins. Thus, according to the current genome annotation methods, a detailed structural/functional characterization at the protein level of each of the elements codified in the genome is also required. The extreme difficulty in obtaining high-resolution three-dimensional structures, calls for computational approaches. Here we review to which extent the efforts made in the last few years, combining the structural characterization of membrane proteins with protein bioinformatics techniques, could help describing membrane proteins at a genome-wide scale. In particular we analyze the use of comparative modeling techniques as a way of overcoming the lack of high-resolution three-dimensional structures in the human membrane proteome.

Dynamic characterization and substrate binding of cis-2,3-dihydrobiphenyl-2,3-diol dehydrogenase-an enzyme used in bioremediation.

In recent years, techniques involving the use of organisms to remove or neutralize pollutants from contaminated sites have attracted great attention. The aim of bioremediation is to use naturally occurring organisms to degrade dangerous substances to less toxic or non toxic molecules. The gram-negative bacterium Pandoraea pnomenusa strain B-356 (Pp) has been found to be able to transform a persistent class of organic pollutant compounds, namely the biphenyl and polychlorinated biphenyls (PCBs). A key enzyme in the PCB catabolic pathway is NAD-dependent cis-2,3-dihydrobiphenyl-2,3-diol dehydrogenase (BphB), for which the crystal structure from Pp has been crystallized in apo-, NAD-bound and biphenyldiol-/NAD-bound forms. The substrate binding loop structure has not been completely resolved to date in the former two bound states. Here we report the results of the first extensive molecular dynamics simulations on the three different states of PpBphB. This allowed an in depth characterization of the mechanism of ligand uptake and binding, including unraveling of the gating mechanism. Our simulations give a deep insight into several dynamic features of the enzyme that were not captured by crystal structures.