O metabolismo de vitamina B9 é um alvo terapêutico conhecido para malária. A vitamina B6 foi validada como essencial para o parasita. Esse trabalho analisa bioquimica- e estruturalmente mutantes da enzima piridoxal quinase, pela combinação de análises in silico associadas a ensaios bioquímicos. A estrutura da PdxK de P. falciparum permitiu a localização do sítio ativo e da interface de dimerização. Logo foram sugeridas mutações que alterassem esses resíduos para investigar sua importância. Dinâmica molecular sugere que a dimerização é responsável pela estabilidade do sítio ativo, algo coerente com a diminuição da atividade enzimática na maioria das mutantes. Filtração em gel aponta um equilíbrio entre a conformação monômero-dímero mostrando que as mutações na interface não estão relacionadas a dimerização, mas envolvidas com a estabilização do folding na região do sítio ativo. A mesma é recoberta por uma tampa que impede a auto-hidrólise do ATP, há também um resíduo serina que estabiliza a conformação do piridoxal durante a catálise, as mutações em ambas as regiões levaram a inativação da enzimática. A hipótese de que a interação da PfPdxK com ligantes de RNA não se mostrou conclusiva. / Vitamin B9 metabolism is a known drug-target for malaria. Vitamin B6 was validated as essential for the parasite. We analysed biochemically and structurally the plasmodial dimeric pyridoxal kinase. PfPdxKs allowed us to determine the localisation of the active site as well the interface between the two monomers and to identify the involved residues. Molecular dynamics shows that the PdxKs dimerization is important for the active sites stability, which was confirmed by the decrease of activity in mutants related to this region. Gel filtration revealed equilibrium of monomer-dimer conformation and therefore the interface mutations decrease in activity might not be directly related to the dimerization processes, but rather to the active site organisation. The active site region shows a serine involved in keeping the pyridoxals conformation during catalyses and the identified lid region covering the ATP binding site is responsible for preventing auto-hydrolysis. Substitution of the respective amino acid to alanine resulted in enzyme inactivation. In silico analysis of the PfPdxK spacer region identified nucleic acid binding sides, however RNA binding experiments failed so far and the possibility of protein-RNA binding remains for elucidation.

No description available.

Metabolism of D-amino acids is of considerable interest due to their key importance in cellular functions. The enzymes D-serine dehydratase (DSD) and D-cysteine desulfhydrase (DCyD) are involved in the degradation of D-Ser and D-Cys, respectively. We determined the crystal structure of Salmonella typhimurium DSD (StDSD) by multiple anomalous dispersion method of phasing using selenomethione incorporated protein crystals. The structure revealed a fold typical of fold type II PLP-dependent enzymes. Although holoenzyme was used for crystallization of both wild type StDSD (WtDSD) and selenomethionine labeled StDSD (SeMetDSD), significant electron density was not observed for the co-factor, indicating that the enzyme has a low affinity for the cofactor under crystallization conditions. Interestingly, unexpected conformational differences were observed between the two structures. The WtDSD was in an open conformation while SeMetDSD, crystallized in the presence of isoserine, was in a closed conformation suggesting that the enzyme is likely to undergo conformational changes upon binding of substrate as observed in other fold type II PLP-dependent enzymes. Electron density corresponding to a plausible sodium ion was found near the active site of the closed but not in the open state of the enzyme. Examination of the active site and substrate modeling suggested that Thr166 may be involved in abstraction of proton from the Cα atom of the substrate. Apart from the physiological reaction, StDSD catalyses α, β-elimination of D-Thr, D-Allothr and L-Ser to the corresponding α-keto acids and ammonia. The structure of StDSD provides a molecular framework necessary for understanding differences in the rate of reaction with these substrates. Salmonella typhimurium DCyD (StDCyD) is a fold type II PLP-dependent enzyme that catalyzes the degradation of D-Cys to H2S and pyruvate. We determined the crystal structure of StDCyD using molecular replacement method in two different crystal forms. The better diffracting crystal form obtained in presence of benzamidine illustrated the influence a small molecule in altering protein interfaces and crystal packing. The polypeptide fold of StDCyD consists of a small domain (residues 48-161) and a large domain (residues 1-47 and 162-328) which resemble other fold type II PLP-dependent enzymes. X-ray crystal structures of StDCyD were also obtained in the presence of substrates, D-Cys and βCDA, and substrate analogs, ACC, D-Ser, L-Ser, D-cycloserine (DCS) and L-cycloserine (LCS). The structures obtained in the presence of D-Cys and βCDA show the product, pyruvate, bound at a site 4.0-6.0 Å away from the active site. ACC forms an external aldimine complex while D and L-Ser bind non-covalently suggesting that the reaction with these ligands is arrested at Cα proton abstraction and transimination steps, respectively. In the active site of StDCyD cocrystallized with DCS or LCS, electron density for a pyridoxamine phosphate (PMP) was observed. Crystals soaked in cocktail containing these ligands show density for PLP-cycloserine. Spectroscopic observations also suggested formation of PMP by the hydrolysis of cycloserines. Mutational studies suggested that Ser78 and Gln77 are key determinants of enzyme specificity and the phenolate of Tyr287 is responsible for Cα proton abstraction from D-Cys. Based on these studies, we proposed a probable mechanism for the degradation of D-Cys by StDCyD. The acid-induced arginine decarboxylase (ADC) is part of an enzymatic system in Salmonella typhimurium that contributes to making this organism acid resistant. ADC is a PLP-dependent enzyme that is active at acidic pH. It consumes a proton in the decarboxylation of arginine to agmatine, and by working in tandem with an arginine-agmatine antiporter, this enzymatic cycle protects the organism by preventing the accumulation of protons inside the cell. We have determined the structure of the acid-induced StADC to 3.1 Å resolution. StADC structure revealed an 800 kDa decamer composed as a pentamer of five homodimers. Each homodimer has an abundance of acidic surface residues, which at neutral pH prevent inactive homodimers from associating into active decamers. Conversely, acidic conditions favor the assembly of active decamers. Therefore, the structure of arginine decarboxylase presents a mechanism by which its activity is modulated by external pH.

<p>Opisane su sinteze, fizičko-hemijske i strukturne karakteristike kompleksa Cu(II), Co(II), Co(III), Ni(II), Fe(III), Cr(III) Zn(II) i Cd(II) sa PLSC, PLTSC i PLITSC. Dobijene su dve nove protonovane forme liganda PLSC, kao i od ranije poznati neutralna forma PLSC i protonovana forma PLTSC ali u vidu monokristala pogodnih za rentgeno-strukturnu analizu i 37 kompleksa, od čega je 12 okarakterisano rentgeno-strukturnom analizom. U zavisnosti od pH sva tri liganda se mogu koordinovati kao neutralni, mono-, odnosno, dianjoni sa uobičajenim ONO, ONS i ONN setom donorskih atoma, respektivno, pri čemu neutralna i monoanjonska forma liganada postoji u obliku zwitter-jona. U slučaju PLTSC a na primerima dva dimerna kompleksa bakra nađen je i ređi tetradentatni ONSO način koordinacije, tj. uče&scaron;će i atoma kiseonika hidroksimetil grupe kao mostovnog atoma. Sva tri liganda sa Cu(II), nezavisno od stehiometrije Cu:ligand, daju mono(ligand) komplekse kvadratno-planarne, kvadratno-piramidalne ili oktaedarske strukture, a sa kobaltom mono- i bis(ligand) oktaedarske komplekse. Dobijenim mono(ligand) kompleksima Zn(II) sa sva tri liganda je pripisana najmanje tetra-, odnosno pentakoordinacija, dok je me&scaron;ovitim PLSC(PLTSC)/NCS i bis(ligand) kompleksima Cr(III), i mono(ligand) kompleksu Fe(III) sa PLTSC pripisuje oktaedarska struktura. Ni(II) sa PLTSC i PLITSC daje mono- i dinuklearne komplekse, dok Cd(II) sa istim ligandima gradi mono(ligand) komplekse. Sva jedinjenja su okarakterisana parcijalnom mikroanalizom, IR-spektrima i konduktometrijskim merenjima, a odabrani kompleksi rentgeno-strukturnom, termogravimetrijskom i toksikolo&scaron;kom analizom.</p> / <p>The work describes the syntheses, physico-chemical, structural and antibacterial characteristics of Cu(II), Co(II), Co(III), Zn(II), Ni(II), Fe(III), Cr(III) and Cd(II) complexes with semi-, thiosemi- and S-methylisothiosemicarbazones. The result of the syntheses was 2 new ligand forms and 37 complexes, of which 12 were characterized by X-ray structural analysis. Depending of the pH, all three ligands can be coordinated either as neutral, monoanion, or dianion, using ONO, ONS and ONN set of donor atoms, respectively, whereby the neutral and monoanionic forms of the ligands exist in the form of zwitter-ions. All three ligands form with Cu(II), irrespective of the stoichiometry Cu:ligand, give mono- and dinuclear complexes of square-planar, square-pyramidal and/or octahedral configuration and with cobalt mono- and bis(ligand) octahedral complexes. The obtained mono(ligand) complexes of Zn (II) with all three of ligands is attributed to the at least tetra-, or pentacoordination, while the mono- and bis(ligand) complexes of Cr(III) with PLSC and PLTSC, and mono (ligand)complex of Fe(III) with PLTSC attributable to octahedral structure. Ni(II) with PLTSC and PLITSC forms mono- and binuclear complexes, and Cd(II) with the same ligands forms mono(ligand) complexes. All the compounds were characterized by partial microanalysis, IR spectra and conductometric measurements, while some selected complexes were examined by X-ray structural, thermogravimetric and toxicological analysis.</p>

Pyridoxal 5’-phosphate (PLP), the active form of vitamin B6, is a cofactor for many enzymes involved in the metabolism of amino acids, amino acid derived metabolites and some amino sugars. PLP is one of the most versatile cofactors and the PLP-dependent enzymes catalyze a variety of reactions including transamination, decarboxylation, inter-conversion of L-and D-amino acids and removal or replacement of chemical groups bound at β or γ carbon of amino acids. The thesis describes the structural studies carried out on three PLP-dependent enzymes; N-acetylornithine aminotransferase (AcOAT), serine hydroxymethyltransferase (SHMT) and diaminopropionate ammonia lyase (DAPAL). Chapter 1 of the thesis begins with a brief introduction to PLP-dependent enzymes and their classification. This is followed by a review of structures of enzymes belonging to the subgroup II aminotransferases. The last section of chapter I contains a detailed description of the structures available till date for SHMT from various sources and the mutational studies carried out on SHMT. All the common experimental procedures and computational methods used for the current investigations are described in chapter II, as most of these are applicable to all structure determinations and analyses. The experimental procedures described include cloning, overexpression, purification, crystallization, and X-ray diffraction data collection. Computational methods include details of various programs used during data processing, structure determination, refinement, model building, structure validation and analysis. AcOAT is one of the key enzymes in arginine and lysine metabolism. AcOAT belongs to the fold type I (αfamily) subgroup II family of PLP dependent enzymes. Both S. typhimurium and E. coli have two genes each, one involved in the biosynthesis of arginine and another in the biodegradation of arginine. Biosynthetic AcOAT catalyzes the conversion of N-acetylglutamate semialdehyde to N-acetylornithine (AcOrn) in the presence of L-glutamate and the conversion of N-succinyl-L-2-amino-6-oxopimelate to N-succinyl-L,L-diaminopimelate in lysine biosynthesis. Meso-DAP and lysine, the products of lysine biosynthesis pathway, are known to function as cross-linking moieties in the peptidoglycan component of bacterial cell wall. Therefore N-acetylornithine aminotransferase could serve as a target for designing antibacterials. Chapter III gives the details of the work carried out on AcOAT. Two genes each from S. typhimurium and E. coli coding for biosynthetic and biodegradative AcOAT were cloned in E. coli, overexpressed and purified by Ni-NTA affinity chromatography. Of the four enzymes, biosynthetic AcOAT from S. typhimurium (sArgD) crystallized in the unliganded form and in the presence of the inhibitor gabaculine or one of the substrates L-glutamate, diffracted to a maximum resolution of 1.90 Å and contained a dimer in the asymmetric unit. The structure was determined by the molecular replacement method using human ornithine aminotransferase (hOAT) as the starting model. The structure of unliganded sAcOAT showed significant electron density for PLP in only one of the subunits (subunit A). The asymmetry in PLP binding could be attributed to the ordering of the loop Lαk-βm in only one subunit. The Km and kcat/Km values determined with the purified sArgD suggested that the enzyme could accept both acetylornithine (AcOrn) and ornithine (Orn) as the substrates and had much higher affinity for AcOrn than for Orn. However, OAT accepts only Orn as the substrate. Comparison of the structurte of sArgD with T. thermophilus AcOAT and hOAT suggested that the higher specificity of sArgD towards AcOrn may not be due to specific differences in the active site residues but could result from minor conformational changes in some of them. sArgD was inhibited by gabaculine with an inhibition constant (Ki) of 7 µM and a second order rate constant (k2) of 0.16 mM-1s-1. The crystal structure of sArgD obtained in the presence of gabaculine and the spectral studies of sArgD with gabaculine suggested that the enzyme might have a low affinity for the PLP-gabaculine complex. Biosynthetic AcOAT from E. coli (eArgD) crystallized in the presence of gabaculine in hanging drop vapor diffusion method and diffracted X-rays only to a resolution of 3.5 Å. Two data sets were collected for the eArgD crystals. One of the data sets belonged to P1 (data 1) and the other to P321 space group (data 2) with a solvent content of ~70%. Data 1 was twinned and the unit cell was unusually large and could accommodate ~24 molecules in the asymmetric unit where as data 2 had four molecules in the asymmetric unit. Biodegradataive AcOAT from E. coli also crystallized in presence of gabaculine in hanging drop vapor diffusion method and suffered from low diffraction quality, where as that from S. typhimurium did not yield crystals. In chapter IV, X-ray crystallographic studies on various site specific mutants of SHMT from Bacillus stereotherophilus (bs) and a detailed comparison of structural data with the biochemical results in relation to mechanism of catalysis are presented. SHMT is a member of the α-class of PLP-dependent enzymes and catalyzes the reversible conversion of L-Ser and THF to glycine and 5,10-methylene THF. 5,10-methylene THF serves as a major source of one-carbon units in the biosynthesis of nucleotides and a few amino acids. SHMT also catalyses the cleavage of β-hydroxy amino acids like L-allo-threonine, transamination, racemization and decarboxylation reactions. SHMT shows increased activity along with enhanced nucleotide synthesis and therefore is a potential target for cancer chemotherapy. The availability of structural and biochemical data on SHMT from different sources ranging from human to E. coli enabled the identification of active site residues and a more critical examination of the role of these residues in the different steps of catalysis. The important mutants studied in the present investigation are E53Q, Y51F, Y61F, Y61A, Y60A, N341A and F351G of bsSHMT. The crystal structures of all these mutants are solved in the presence of various ligands, which gave many interesting results. E53, one of the active residues, interacts with the side chain hydroxyl group of serine bound to PLP in the wild type serine complex and N10 and formyl oxygen in the wild type glycine-FTHF complex. In E53Q glycine and serine complexes, glycine carboxyl and serine side chain were in two conformations, respectively, the new conformation being stabilized by their interaction with the mutated residue Q53. The structure of E53Q-Gly complex obtained in the presence and absence of 5-formyl THF(FTHF) showed an interesting case of enzyme memory in which the final conformational state depends on the way it was obtained and suggested that E53 is crucial for FTHF/THF binding. Though the spectrum showed that FTHF binds to the mutant initially, no density was observed for FTHF in the final structure. FTHF is believed to dissociate from the active site with prolonged incubation leaving behind a few significant conformational changes. Y51, one of the highly conserved tyrosines in SHMT, has hydrogen bonding interactions with the phosphate group of PLP and the active site lysine (K226) in bsSHMT. Mutation of Y51 to F resulted in significant changes at the active site. In all the structures of Y51F complexes, the phosphate group is in two conformations and F51 has moved away from the phosphate and in turn changed the position of Y61, another tyrosine in the active site. The residue Y61 is hydrogen bonded to R357 in the internal aldimine complex of bsSHMT. Addition of glycine/serine to bsSHMT resulted in the conformational change of Y61 away from R357 and towards E53, allowing the added glycine/serine to interact with R357. Mutation of Y61 to A did not bring significant structural changes. Structures of Y51F and Y61A mutants complexed with L-allo-Thr (cleaved to Gly by the wild type enzyme) showed that L-allo-Thr was not cleaved to glycine and acetaldehyde and confirmed the biochemical observation that these two residues are essential even for the THF-independent reaction. Residues Y60 and N341 are also highly conserved residues among SHMTs. Y60 stacks over PABA ring of FTHF in the wild type glycine-FTHF ternary complex. N341 has strong hydrogen bonding interactions with N1 and N8 atoms of the pteridine ring of FTHF. Mutation of either Y60 or N341 to A destroys the binding ability of FTHF/THF to the enzyme according to the biochemical and structural observations. The residue F351 exhibits different conformations in the two subunits of wild type glycine-FTHF ternary complex and is thought to be an important residue in determining the asymmetric binding of FTHF. Mutation of F351 to G did not affect the catalytic activity. Surprisingly, in the crystal structure obtained in the presence of L-allo-Thr, the ligand did not get cleaved to glycine, though in solution, the mutant is as active as the wild type enzyme. Chapter V describes the preliminary structural studies carried out on DAPAL from E. coli and S. typhimurium. DAPAL catalyzes the α, βelimination of both L-and D-diaminopropionate (DAP). DAP is the immediate precursor of two neurotoxins 3oxalyl and 2,3-dioxalyl DAP present in Lathyrus sativus, a grain legume rich in proteins and capable of growing well in drought conditions. The presence of these two neurotoxins precludes its use as a source of protein rich food. This enzyme is present only in bacteria and few species of actinomycetes. Unlike many other PLP-dependent enzymes, DAPAL does not catalyze any side reaction and is the only enzyme known to remove an amino group from the βcarbon of the substrate. The enzymes from E. coli (eDAPAL) and S. typhimurium (sDAPAL) produced diffraction quality crystals. However, crystals of sDAPAL did not survive heavy atom soaking and eDAPAL crystals suffered from poor reproducibility and severe non-isomorphism making it difficult to obtain suitable heavy atom derivatives for structure determination. Production of selenomethionine labelled proteins for these enzymes was initiated and thin crystals were obtained for eDAPAL. Improvement of the quality of these crystals is necessary in order to solve the structure of DAPAL by MAD method.

No description available.

No description available.

ABSTRACT The porphyrias are a diverse group of metabolic disorders arising from diminished activity of enzymes in the heme biosynthetic pathway. They can present with acute neurovisceral symptoms, cutaneous symptoms, or both. The complexity of these disorders is demonstrated by the fact that some acute porphyria patients with the underlying genetic defect(s) are latent and asymptomatic while others present with severe symptoms. This indicates that there is at least one other risk factor required in addition to the genetic defect for symptom manifestation. A systematic review of the heme biosynthetic pathway highlighted the involvement of a number of micronutrient cofactors. An exhaustive review of the medical literature uncovered numerous reports of micronutrient deficiencies in the porphyrias as well as successful case reports of treatments with micronutrients. Many micronutrient deficiencies present with symptoms similar to those in porphyria, in particular vitamin B6. It is hypothesized that a vitamin B6 deficiency and related micronutrient deficiencies may play a major role in the pathogenesis of the acute porphyrias. In order to further investigate the porphyrias, a computational model of the heme biosynthetic pathway was developed based on kinetic parameters derived from a careful analysis of the literature. This model demonstrated aspects of normal heme biosynthesis and illustrated some of the disordered biochemistry of acute intermittent porphyria (AIP). The testing of this model highlighted the modifications necessary to develop a more comprehensive model with the potential to investigated hypotheses of the disordered biochemistry of the porphyrias as well as the discovery of new methods of treatment and symptom control. It is concluded that vitamin B6 deficiency might be the risk factor necessary in conjunction with the genetic defect to trigger porphyria symptoms.