Agmatine, an endogenous amine derived from decarboxylation of l-arginine catalyzed by arginine decarboxylase, has been proposed as a neurotransmitter or neuromodulator in the brain. In the present study, we examined whether agmatine has neuroprotective effects against repeated immobilization-induced morphological changes in brain tissues and possible effects of immobilization stress on endogenous agmatine levels and arginine decarboxylase expression in rat brains. Sprague-Dawley rats were subjected to 2 h immobilization stress daily for 7 days. This paradigm significantly increased plasma corticosterone levels, and the glutamate efflux in the hippocampus as measured by in vivo microdialysis. Immunohistochemical staining with β-tubulin III showed that repeated immobilization caused marked morphological alterations in the hippocampus and medial prefrontal cortex that were prevented by simultaneous treatment with agmatine (50 mg/kg/day), i.p.). Likewise, endogenous agmatine levels measured by high-performance liquid chromatography in the prefrontal cortex, hippocampus, striatum and hypothalamus were significantly increased by immobilization, as compared to controls. The increased endogenous agmatine levels, ranging from 92 to 265% of controls, were accompanied by a significant increase of arginine decarboxylase protein levels in the same regions. These results demonstrate that the administration of exogenous agmatine protects the hippocampus and medial prefrontal cortex against neuronal insults caused by repeated immobilization. The parallel increase in endogenous brain agmatine and arginine decarboxylase protein levels triggered by repeated immobilization indicates that the endogenous agmatine system may play an important role in adaptation to stress as a potential neuronal self-protection mechanism.

Most of the chemical reactions of living cells are catalyzed by protein enzymes. These enzymes are very efficient and display a high degree of specificity with respect to the reaction catalyzed. Cellular activities depend critically on the precise three-dimensional structure and function of thousands of enzymes. Many enzymes require binding of metal ions or small organic molecules for their function. The organic molecules that are indispensible components of catalysis by proteins are called coenzymes. Pyridoxal 5ʹ-phosphate (PLP) is a versatile coenzyme found in all living cells. PLP-dependent enzymes play a key role in the function of most of the enzymes catalyzing reactions in the metabolic pathways of amino acid synthesis and degradation. The enzyme pyridoxal kinase serves to make available the co-enzyme PLP to apo-PLP dependent enzymes. Because of their key role in cellular function and their medical importance, the structure and function of PLP-dependent enzymes have been extensively investigated. In the past decade, detailed investigations on the structure and function of several PLP-dependent enzymes have been carried out in our laboratory. The enzymes studied are B. subtilis serinehydroxymethyl transferase (SHMT), S. typhimurium acetylornithine aminotransferase (AcOAT), S. typhimurium and E. coli diaminopropionate ammonia lyase (DAPAL), S. typhimurium D-serine dehydratase (DSD), S. typhimurium D-cysteine desulfhydrase (DCyD) and S. typhimurium arginine decarboxylase (ArgD). The extensive studies conducted on PLP-dependent enzymes in our laboratory during the past decade has not only resulted in deeper understanding of their structure and function but also raised several new questions regarding substrate recognition, reaction specificity, role of active site residues in the catalytic reaction, mechanism of catalysis and potential applications of these enzymes. This thesis is an attempt to answer some of these questions. The thesis also presents the structure and function of a new protein, Salmonella typhimurium pyridoxal kinase, the enzyme that provides PLP for PLP-dependent enzymes. Single crystal X-ray diffraction technique is the most powerful tool currently available for the elucidation of the three-dimensional structures of proteins and other biological macromolecules and for revealing the relationship between their structure and function. X-ray diffraction studies have provided in depth understanding of the topology of secondary structural elements in the three-dimensional structures of proteins, the hierarchical organization of protein domains, structural basis for the substrate specificity of enzymes, intricate details of mechanisms of enzyme catalyzed reactions, allosteric regulation of enzyme activity, mechanisms of feed-back inhibition, structural basis of protein stability, symmetry of oligomeric proteins and their possible biological implications and a myriad of other biochemical and biophysical properties of proteins. The work reported in this thesis is primarily based on X-ray diffraction studies. X-ray crystal structure investigations are complemented by spectral and biochemical studies on the catalyzed reactions. The thesis begins with an introduction to PLP-dependent enzymes and presentation of a brief summary of the earlier work carried out in our laboratory on PLP-dependent enzymes (Chapter 1). A brief description of earlier functional classification of PLP-dependent enzymes and the more recent classification of these enzymes into the four groups based on their three-dimensional structure is provided. Although enzymes belonging to these four structural classes have evolved from independent evolutionary lineages, they share some common features near their active sites and in the mode of PLP binding. Earlier work carried out elsewhere on pyridoxal kinase and its key role in maintaining PLP at a low concentration in the cytosol is presented. Different mechanisms that have been proposed for the transfer of PLP from pyridoxal kinase to other apo PLP-dependent enzymes are briefly described. The experimental procedures and computational methods used during the course of these investigations to obtain the results reported in chapters 3-6 are presented in Chapter 2. Most of these methods are applicable to the isolation of plasmids, cloning, over expression, protein purification, mutant construction, crystallization, X-ray diffraction data collection and processing, structure elucidation and refinement, validation and structural analysis presented in the next three chapters. Various programs and protocols used for data processing, structure determination, refinement, model building, structure validation and analysis are also briefly described. In chapter 3, the role of a number of active site residues in the reaction catalyzed by EcDAPAL, a fold type II PLP-dependent enzyme, the structure of which was determined earlier in the laboratory is explored by mutational, biochemical and structural analyses. Earlier studies had established the probable role of Asp120 and Lys77 in the reaction leading to the breakdown of D-DAP and L-DAP, respectively (Bisht et al., 2012). To further validate the earlier observations, a number of active site mutants were generated for Asp 120 (D120N, D120C, D120S and D120T), Asp 189 (D189N, D189C, D189S and D189T), Lys77 (K77T, K77H, K77R and K77A), His 123 (H123L) and Tyr 168 (Y168F). The structure of D120N mutant crystal obtained after soaking in crystallization cocktail containing D-DAP revealed the presence of an intact external aldimine complex at the active site supporting the earlier proposal that Asp120 is the base abstracting the Cα proton from the D-isomer of DAP. Biochemical and structural observations suggested that none of the Asp189 mutants may bind PLP and were catalytically inactive suggesting an essential role for Asp189 in catalysis. In contrast to type I PLP-dependent enzymes, none of the Lys 77 mutants of EcDAPAL could bind PLP either covalently or non-covalently and were inactive with both the isomers of DAP. Thus, Lys77 appears to be important for both PLP binding and catalysis. H123L mutant formed an external aldimine with D-DAP and a gem-diamine complex with L-DAP indicating that this residue is also crucial for catalysis. These studies have provided additional support to the catalytic mechanism of EcDAPAL proposed earlier. The next Chapter 4 explores the structure, function and catalytic mechanism of Salmonella typhimurium DAPAL (StDAPAL). The protein was purified from a construct carrying a hexa-histidine tag at the C-terminus by Ni-NTA chromatography. The purified protein was demonstrated to be homogeneous by SDS-PAGE and MALDI-TOF. Crystals of StDAPAL belonging to the C-centred monoclinic space group (C121) with four molecules in the asymmetric unit were obtained by the micro batch method and used for collecting X-ray diffracting data. The crystal structure was determined by molecular replacement using the homologous enzyme from E. coli (PDB code 4D9M, Bisht et al., 2012), which shares a sequence identity of 50% with the S. typhimurium enzyme as the phasing model in the program Phaser (McCoy et al., 2007) of the CCP4 suite. The model was refined with Refmac5 of CCP4 suite to R and Rfree values of 25.5% and 30.9%, respectively. A superposition of the structure so obtained over EcDAPAL revealed that the two structures are very similar. A sulfate molecule bound to the active site of StDAPAL could be located. The position of the sulfate corresponds to that of the carboxyl group of aminoacrylate intermediate of EcDAPAL (4D9M). The PLP was bound to Lys78 as an internal aldimine. Since the active sites of the two protomers in fold type II PLP-dependent enzymes are independent, it might be possible to obtain functional monomers of EcDAPAL. With this view, mutation of a conserved Trp (Trp399) present in the dimeric interface resulted in the destabilization of the dimeric interface and partial conversion of the dimeric protein to a monomeric protein. However, the monomeric species of EcDAPALW399R was unable to bind PLP and hence did not possess any catalytic activity. This highlights the importance of dimeric organization for efficient binding of PLP as well as for the activity of the enzyme. A remarkable difference between EcDAPAL and StDAPAL is the absence of a disulfide bond between residues Cys271 and Cys299 in StDAPAL equivalent to the bond formed between Cys265 and Cys291 in EcDAPAL. Mutation of Cys265 and Cys291 of EcDAPAL to Ser did not affect the activity of the enzyme towards either of the isomers of the substrate indicating that the disulfide bond is not crucial for enzyme activity. The stability of the loop corresponding residues 261-295 of EcDAPAL was believed to be promoted by the disulfide bond. However, the equivalent loop was found to be ordered in StDAPAL even though the disulfide bond is absent. In contrast to StDAPAL, EcDAPAL did not show any metal dependent activity. The previous two chapters dealt with fold type II PLP-dependent enzymes. In contrast, Chapter 5 deals with revisiting the structure and function of a fold type I PLP-dependent enzyme, Salmonella typhimurium arginine decarboxylase (StADC). ADC is a very large polypeptide in comparison with other fold type I enzymes. It is induced when the bacterium is subjected to low pH and plays a major role in protecting the cells from acid stress. The structure of StADC was determined but not satisfactorily refined by Dr. S. R. Bharat earlier. The X-ray diffraction data collected by Bharat needed to be improved and the structure needed to be further refined and compared with the homologous E. coli enzyme. Therefore, the entire process of data processing, structure solution and refinement was repeated. The refined structure of StADC was found to correspond to the apo form of the enzyme with only a phosphate molecule occupying the position equivalent to that of 5’ phosphate of PLP observed in EcADC holo enzyme structure. This allowed examination of structural changes that accompany PLP binding and formation of an internal aldimine. The apo to holo transition in StADC involves the movement and ordering of two loops consisting of residues 151-164 and 191-196 which are in the linker and PLP binding domains of the protein, respectively. Phosphate binding by itself appears to be insufficient for these structural changes. These two loops are close to the PLP binding site of the other protomer of the dimer. Hence, these movements are probably important for the catalytic function of the enzyme. Holo ADC has been found as a decamer in other studies. The decameric form of the apo-StADC suggests that PLP binding may not be essential for the oligomeric state of the protein. ADC appears to reduce proton concentration inside the cell in two ways; (i) by surface charge neutralization and (ii) by arginine decarboxylation by extracting a proton from the cytoplasm. The resulting product agmatine is exchanged for extra cellular arginine by arginine-agmatine antiporter. The low sequence identity and lack of structural similarity of the inducible and constitutive forms of ADC from S. typhimurium shows that these are unlikely to be products of divergent evolution. The final chapter 6 of the thesis presents the work carried out on S. typhimurium pyridoxal kinase (PLK). In the salvage pathway of pyridoxal 5’phosphate (PLP), PLP is produced as the product of the reaction catalyzed by PLK using PL, PN and PM as substrates. Thus, PLK plays the critical role of ensuring availability of PLP to the large number of PLP-dependent enzymes. S. typhimurium PLK was purified to homogeneity, crystallized in its native as well as ligand bound forms. It was necessary to circumvent an unusual problem caused by spots arising from a contaminant crystal to obtain the structure of the native crystals of PLK that belonged to the P212121 space group with two protomers in the crystal asymmetric unit. It was then straight forward to determine the ligand bound structures of StPLK (space group P43212) obtained by co-crystallization with ATP, PL and Mg2+ by molecular replacement using the wild type structure as the phasing model. The structures obtained by co-crystallization revealed the presence of ADP, Mg2+ and a PL bound to the active site Lys233 via a Schiff base (internal aldimine). This is the first structure in which the presence of an internal aldimine in the active site of PLK has been observed. Formation of the internal aldimine might be one way to prevent the release of excess PLP and protecting the cell from PLP induced toxicity. The enzyme was shown to be inhibited by the product which will also help in maintaining PLP concentration at low levels. It was also demonstrated that PLK interacts with apo-PLP-dependent enzymes. This observation supports possible direct transfer of PLP from PLK to PLP-dependent enzymes. The thesis ends with an appendix where the work carried out during the course of the thesis work but not as part of the thesis is briefly described.

The nature of the subunit interactions in the inducible arginine decarboxylase from Escherichia coli B is of considerable interest because of the observed differences in the catalytic activities of the dimer and the decamer; the decamer is active and the dimer is inactive. To study these interactions, inactive dimers were prepared by sodium borohydride reduction of the E-amino--pyridoxal-P Schiff base. Hybrid decamers were then prepared from varying molar ratios of native and reduced dimers. The hybrid decamers were indistinguishable from native decamers as observed in the analytical ultracentrifuge and on acrylamide gel electrophoresis. Kinetic studies indicated that true hybrids were formed rather than mixtures of all-native and all-reduced decamers. Results obtained with the decamers containing 1, 2, 3, or 4 parts in 5 of reduced enzyme showed no significant changes in Km values from the native decamer. However, the Vm values for these hybrids are greater than predicted from the mole fraction of active dimers. For example, the hybrid containing 20% reduced enzyme approaches the Vm of the native decamer. These observations suggest that, in the intact molecule, two active sites cooperate catalytically but only one is catalytically active.

Agmatine is an endogenous amine derived from decarboxylation of arginine catalysed by arginine decarboxylase. Agmatine is considered a novel neuromodulator and possesses neuroprotective properties in the central nervous system. The present study examined whether agmatine has neuroprotective effects against repeated restraint stress-induced morphological changes in rat medial prefrontal cortex and hippocampus. Sprague-Dawley rats were subjected to 6 h of restraint stress daily for 21 days. Immunohistochemical staining with β-tubulin III showed that repeated restraint stress caused marked morphological alterations in the medial prefrontal cortex and hippocampus. Stress-induced alterations were prevented by simultaneous treatment with agmatine (50 mg/kg/day, i.p.). Interestingly, endogenous agmatine levels, as measured by high-performance liquid chromatography, in the prefrontal cortex and hippocampus as well as in the striatum and hypothalamus of repeated restraint rats were significantly reduced as compared with the controls. Reduced endogenous agmatine levels in repeated restraint animals were accompanied by a significant increase of arginine decarboxylase protein levels in the same regions. Moreover, administration of exogenous agmatine to restrained rats abolished increases of arginine decarboxylase protein levels. Taken together, these results demonstrate that exogenously administered agmatine has neuroprotective effects against repeated restraint-induced structural changes in the medial prefrontal cortex and hippocampus. These findings indicate that stress-induced reductions in endogenous agmatine levels in the rat brain may play a permissive role in neuronal pathology induced by repeated restraint stress.

In the present study, we examined the possible effect of chronic treatment with glucocorticoids on the morphology of the rat brain and levels of endogenous agmatine and arginine decarboxylase (ADC) protein, the enzyme essential for agmatine synthesis. Seven-day treatment with dexamethasone, at a dose (10 and 50 μg/kg/day) associated to stress effects contributed by glucocorticoids, did not result in obvious morphologic changes in the medial prefrontal cortex and hippocampus, as measured by immunocytochemical staining with β-tubulin III. However, 21-day treatment (50 μg/kg/day) produced noticeable structural changes such as the diminution and disarrangement of dendrites and neurons in these areas. Simultaneous treatment with agmatine (50 mg/kg/day) prevented these morphological changes. Further measurement with HPLC showed that endogenous agmatine levels in the prefrontal cortex and hippocampus were significantly increased after 7-day treatments with dexamethasone in a dose-dependent manner. On the contrary, 21-day treatment with glucocorticoids robustly reduced agmatine levels in these regions. The treatment-caused biphasic alterations of endogenous agmatine levels were also seen in the striatum and hypothalamus. Interestingly, treatment with glucocorticoids resulted in a similar change of ADC protein levels in most brain areas to endogenous agmatine levels: an increase after 7-day treatment versus a reduction after 21-day treatment. These results demonstrated that agmatine has neuroprotective effects against structural alterations caused by glucocorticoids in vivo. The parallel alterations in the endogenous agmatine levels and ADC expression in the brain after treatment with glucocorticoids indicate the possible regulatory effect of these stress hormones on the synthesis and metabolism of agmatine in vivo.

This dissertation is a compilation of published work and a manuscript that seeks to understand the role of polyamine metabolism in the regulation of pneumococcal physiology. Streptococcus pneumoniae (pneumococcus) is the major cause of community-acquired pneumonia, and otitis media worldwide. Genetic diversity and serotype replacement, and antibiotics resistance to confound existing therapeutic strategies and limit the effectiveness of the available capsule polysaccharide (CPS) based vaccines. Polyamines such as putrescine, spermidine and cadaverine are ubiquitous polycationic hydrocarbons that interact with negatively charged molecules and modulate important cellular processes. Intracellular polyamine concentrations are regulated by biosynthesis, degradation, and transport. This work investigated the impact of the deletion of polyamine biosynthesis gene, SP_0916 (cadA, lysine/arginine decarboxylase covered in the second, third and fourth chapters), on growth, Gram staining characteristics, capsule production, proteome and stress responses of virulent pneumococcal serotype 4 (TIGR4). We identified loss of capsular polysaccharide (CPS) in DELTA SP_0916 strain. Our proteome results showed a shift in metabolism towards the pentose phosphate pathway (PPP) that could reduce the availability of precursors for CPS and could explain the un-encapsulated phenotype of DELTA SP_0916. Since a shift towards the PPP is usually in response to stress, we compared the stress responses of DELTA SP_0916 to that of TIGR4. Our results show that the mutant was more susceptible to oxidative, nitrosative, and acid stress compared to the wild type. In the fifth chapter we compared the transcriptome, metabolome, stress responses and stress susceptibility of the polyamine transport deficient strain (DELTA potABCD) and S. pneumoniae TIGR4. Results in this chapter show that polyamine transport is essential for pneumococcal stress responses, and capsule biosynthesis. The impact of impaired polyamine synthesis (DELTA SP_0916), and transport (DELTA potABCD) on pneumococcal capsule is due to altered expression of Leloir pathway, reduced glycolysis, and increased PPP, possibly in response to impaired stress responses. These results demonstrate that alteration of polyamine pathways affects pneumococcal stress responses which in turn could limit the availability of precursors for capsule synthesis, and thus have an impact on virulence. Thus, polyamine metabolism is an attractive avenue for developing novel interventions for limiting the spread of S. pneumoniae, a versatile human pathogen.

A Araucaria angustifolia é uma conífera nativa do Brasil. Em função da sua intensa exploração florestal, a espécie ocupa apenas 2% de sua vegetação natural. Neste sistema, a aplicação de técnicas biotecnológicas, como a embriogênese somática, podem ser integradas a programas de melhoramento genético e conservação. A similaridade entre a embriogênese somática e zigótica, tem sido utilizada para o estabelecimento de estudos visando o aperfeiçoamento do cultivo in vitro dos embriões somáticos, bem como para um maior conhecimento dos aspectos moleculares e fisiológicos que regulam a embriogênese. O metabolismo de poliaminas (PAs), mais especificamente putrescina, espermidina e espermina, tem se mostrado como fundamental para a compreensão e evolução da embriogênese zigótica e somática. Entretanto, a biossíntese das PAs e seu envolvimento nos vários processos biológicos que regulam a embriogênese, são pouco conhecidas em coníferas. Inserido nessa perspectiva, o presente trabalho teve como objetivo o estudo do metabolismo de PAs durante três estádios de desenvolvimento da semente (contendo as fases da embriogênese inicial até a tardia) e na proliferação de linhagens embriogênicas com diferentes potenciais embriogênicos de A. angustifolia. Foram investigados: a) os perfis de PAs (livres e conjugadas) e aminoácidos; b) determinação da via preferencial da biossíntese de putrescina, através da atividade enzimática da arginina descarboxilase (ADC) e ornitina descarboxilase (ODC); c) identificação e caracterização do padrão de expressão dos genes envolvidos no metabolismo de PAs; e d) a identificação das relações entre os perfis de PAs e aminoácidos presentes nas sementes das matrizes, e sua potencial influência nas fases de indução, proliferação e maturação dos embriões somáticos. Durante a embriogênese zigótica, a expressão dos genes AaADC (arginina descarboxilase) e AaSAMDC (S-adenosilmetionina descarboxilase) aumentaram no estádio cotiledonar, juntamente com o aumento de PAs. A biossíntese da putrescina é realizada preferencialmente via ADC, enquanto que a citrulina foi o principal aminoácido presente nas sementes. Em relação ao metabolismo de PAs nas culturas embriogênicas, os dados obtidos demonstraram que a arginina e ornitina parecem ter diferentes funções em cada linhagem testada. Na linhagem com alto potencial embriogênico, a arginina parece estar associada com a ativação dos genes relacionados ao catabolismo de PAs (AaPAO2, AaCuAO e AaALDH), enquanto que esse efeito não foi observado na linhagem bloqueada. A ODC tem uma maior atividade na linhagem responsiva, enquanto que na linhagem bloqueada, as atividades da ADC e ODC são similares. Dependendo da matriz foram observados diferentes perfis de PAs e aminoácidos, sendo estes perfis relacionados com as taxas de indução, proliferação e desenvolvimento dos embriões somáticos. Putrescina total, ornitina e asparagina foram os metabólitos diferencialmente identificados entre as matrizes, os quais podem ser propostos como marcadores bioquímicos para a seleção de matrizes com alto potencial para a embriogênese somática. Os resultados obtidos fornecem informações relevantes e inéditas sobre o metabolismo de PAs e aminoácidos na embriogênese zigótica e somática de A. angustifolia, bem como fornece novos subsídios para o aprimoramento das condições artificiais utilizadas para o desenvolvimento dos embriões somáticos / The Araucaria angustifolia is a native conifer species of Brazil. Due to its intense exploitation, the species cover only 2% of its original forest area. In this system, biotechnological tools, like somatic embryogenesis, may be integrated into breeding and conservation programs. The similarity between zygotic and somatic embryogenesis have been used to establishment of studies in order to optimization of somatic embryos in vitro culture, as well as for a better understanding of physiologic and molecular aspects that modulates the embryogenesis. The metabolism of polyamines (PAs), specifically putrescine, spermidine and spermine, has been demonstrated as fundamental for the comprehension and evolution of zygotic and somatic embryogenesis. However, the biosynthetic pathways of PAs and their involvement in various biological process that regulate the embryogenesis are little known in conifers. Inserted in this perspective, the aim of the current work was to study the metabolism of PAs during three seeds development stages (containing the early till late embryogenesis phases) and in proliferation of cell lines with different embryogenic potential of A. angustifolia. Were investigated: a) PAs (free and conjugated) and amino acids profiles; b) determination of preferential pathway for putrescine biosynthesis, through enzymatic activity of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC); c) identification and characterization of gene expression profile of genes related to metabolism of PAs; and d) identification of the relationship between PAs and amino acids profiles in seeds of mother plants, and their potential influence in initiation, proliferation and maturation phases of somatic embryos. During the zygotic embryogenesis, AaADC (arginine decarboxylase) and AaSAMDC (S-adenosylmethionine decarboxylase) genes were up-regulated at cotyledonary stage along with the increasing of PAs. The biosynthesis of putrescine is performed preferentially by ADC pathway, while citrulline was the main amino acid recorded during the seed development. Regarding the metabolism of PAs in embryogenic cultures, the data demonstrated that arginine and ornithine seem to have different functions in each cell line tested. In cell line with high embryogenic potential, arginine seems to be associated to activation of genes related to PAs catabolism (AaPAO2, AaCuAO e AaALDH), while in blocked cell line this effect was not observed. ODC has a higher enzymatic activity in responsive cell line, while in blocked cell line, both ADC and ODC activities are similar. Depending of mother plant, were observed different PAs and amino acids profiles, being these profiles related with the rate of initiation, proliferation and maturation of somatic embryos. Total putrescine, ornithine and asparagine were the differentially metabolites identified between the mother plants, which can be proposed as biochemical marker to select mother plant with high potential to somatic embryogenesis. The results obtained provide relevant and inedited information about the metabolism of PAs and amino acids in zygotic and somatic embryogenesis of A. angustifolia, as well as provide news subsidies for optimization of in vitro conditions for somatic embryos development

Arginine decarboxylase is a key enzyme involved in the polyamine pathway of organisms. Pyruvoyl-dependent arginine decarboxylases are expressed in the form of proenzymes that self-cleave to form N-terminal [beta] and C-terminal [alpha] subunits generating an active pyruvoyl group at the [alpha] terminus. We have identified an archaeal homolog of a pyruvoyl-dependent arginine decarboxylase in Chlamydophila pneumoniae that could play a role in the persistence of the organism in the host. The recombinant enzyme showed highest activity at pH 3.4, which is the lowest optimum pH ever reported for a pyruvoyl dependent arginine decarboxylase. The proton-consuming decarboxylation raises intracellular pH, and thereby plays a role in acid-resistance. It could inhibit the pro-inflammatory nitric oxide synthase resulting in asymptomatic infection. A variant protein Thr⁵²Ser at the predicted cleavage site showed less pro-enzyme cleavage and activity compared to the wild-type. The homologs of arginine decarboxylase and flanking arginine-agmatine antiporter were also found in different biovariants of Chlamydia trachomatis. In the invasive L2 strain of C. trachomatis, the presence of a nonsense codon in the gene encoding arginine decarboxylase enzyme prevented the expression of an active enzyme. The variant protein with tryptophan replacing nonsense codon restored arginine decarboxylase activity. The non-invasive D strain of C. trachomatis had an intact arginine decarboxylase gene, but it was recombinantly expressed as a proenzyme that was uncleaved. The arginine-agmatine antiporters from both the strains were active and transported tritiated arginine into their cells. The polyamine pathway of the crenarchaeon Sulfolobus solfataricus uses arginine to make putrescine, but the organism lacks homologs of arginine decarboxylase. However, it has two paralogs of pyruvoyl dependent S-adenosylmethionine decarboxylase − SSO0536 and SSO0585. These enzymes were recombinantly expressed as pro-enzymes that self-cleaved into [beta] and [alpha] subunits. Even with a 47% amino acid sequence identity, the SSO0536 protein exhibited significant arginine decarboxylase activity whereas SSO0585 protein had significant S-adenosylmethionine decarboxylase activity. This is the first report of an S-adenosylmethionine decarboxylase enzyme showing alternative decarboxylase activity. The chimeric protein with the [alpha]-subunit of SSO0585 and [beta]-subunit of SSO0536 had arginine decarboxylase activity, suggesting that the residues responsible for substrate recognition are located in the amino terminus. / text

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.