Structural Insight Into The Catalytic Mechanistic Of Yeast Pyridoxal 5-phosphate Synthase Snz1 And Autophagy Pathways Of Bombyx Mori

(I) The active form of vitamin B6, pyridoxal 5-phosphate (PLP), is an essential cofactor for numerous metabolic enzymes and plays an important role in amino acid and carbohydrate metabolism. Up to date, two different pathways for the de novo biosynthesis of PLP have been characterized:deoxyxylulose 5-phosphate (DXP)-dependent and independent pathways. The first pathway, consisting of the pdx family (Pdx A, B, C, F, H, J, and Gap A), is found in a small number of eubacteria and has been extensively studied in Escherichia coli. In this pathway, PLP is derived from 4-phosphohydroxy-L-threonine and DXP. The second pathway, referred to as DXP-independent, is predominant among most eubacteria, fungi, apicomplexa, plants, and even some metazoans. This pathway involves the interaction of two proteins, Pdxl (synthase) and Pdx2 (glutaminase). In this pathway, PLP is formed from ribulose 5-phosphate (or ribose 5-phosphate), glyceraldehyde 3-phosphate (or dihydroxyacetone phosphate) and ammonia formed by the hydrolysis of glutamine.It has been reported that in the presence of ammonia, the synthase Pdxl alone can catalyze a set of reactions including pentose and triose isomerization, imine formation, ammonia addition, and aromatic ring formation. During the reaction sequence of Pdxl, it has been hypothesized that there is a pentulose phosphate imine adduct. Lys149 of B. subtilis Pdx1 was previously identified to be involved in adduct formation with RBP. However, crystallographic evidence has verified that the pentulose phosphate mine occurs at lysine 81 rather than lysine 149. Acordding to enzyme assay result, both K81A and K149A mutants of the B. subtilis Pdxl were unable to catalyze the formation of PLP. In addition, the K80A mutant lost its ability to isomerize R5P to RBP, while the K149A mutant catalyzed this isomerization faster than native enzyme. These results demonstrated that both of these lysine residues plays an essential role in PLP formation and K80 most probably catalyzes the formation of the RBP imine from R5P. However, the other substrates binding sites, the triose isomerization site and PLP formation site remained unclear.The yeast genome harbors three PDX1 paralogs, named SNZ1, SNZ2 and SNZ3, each of which is flanked by a PDX2 gene of SNO1, SNO2, or SNO3 at the complementary chain. SNZ2/SNO2 and SNZ3/SNO3 are involved in vitamin B1 synthesis, while SNZ1/SNO1 are essential for the de novo biosynthesis of PLP. Here we present the crystal structures of PLP synthase Snzl from Saccharomyces cerevisiae, in apo-, G3P-and PLP-bound forms, at 2.30,1.80 and 2.20 A, respectively. Activity assays in combination with site-directed mutagenesis ambiguously confirm the identification of two G3P binding sites and the PLP synthesis site. Structural and biochemical analysis enabled us to assign the PLP-formation site, a G3P-binding site and a G3P-transfer site. We propose a putative catalytic mechanism for Pdx1/Snz1 in which R5P and DHAP are isomerized at two distinct sites and transferred along well-defined routes to a final destination for PLP synthesis. (â…¡) The programmed cell death (PCD) is a genetically regulated program of cell elimination, which is evolutionarily conserved in eukaryotes and plays a very important role in several physiological processes. PCD consists of two major types, apoptosis (typeâ… ) and autophagic cell death (typeâ…¡). For a long time, autophagy has been described as a form of typeâ…¡programmed cell death characterized by lysosomal activation and formation of autophagosomes. It is ubiquitous among eukaryotes functioning as a lysosome degradation pathway for recycling cytoplasmic materials especially long-lived proteins.The formation of autophagosomes depends on the two ubiquitin-like conjugation systems, Atg8-PE (phosphatidylethanolamine) and Atg12-Atg5-Atg16 systems. There are several major signal transduction pathways and complexes involved in the induction of autophagy, including the TOR (target of rapamycin) signal transduction pathway and PI3K-I(PtdIns 3-kinase class I)/Akt(Protien Kinase B) pathway. TOR exerts an inhibitory effect on autophagy through various downstream effectors to regulate the transcription and/or translation of other related genes; the.activation of PI3K-I/Akt pathway inhibits the GTPase-activating activity of Tsc2, leading to the relief of the inhibitory effect of Rheb-GDP on TOR, and down regulate the autophagy level; while PI3K-III, another member of PI3K family, together with its membrane adaptor and autophagy protein Atg6 (Beclin1 in mammals), functions as an activator of autophagy and plays a crucial role in the early steps of autophagosome formation.Recently, an explosion of studies on autophagy and cell survival indicates that autophagy may play an important role in the life cycle of eukaryotic organism. Autophagy may help cells to survive in death mutants, to crosstalk with the regulation of cell proliferation, to remove toxic cytoplasmic constituents, to reduce neurotoxicity of polyglutamine expansion proteins in some neurodegenerative diseases and also to be required for the metamorphic development of insects.The metamorphic development from larvae to pupa is accompanying with the degeneration of specific larval tissues, such as the salivary glands of Drosophila melanogaster, prothoracic glands and silkglands of Bombyx mori. The silkgland is the largest tissue in the fifth and last instar of the silkworm B. mori. It consists of three parts:anterior (ASG), middle (MSG) and posterior silkglands (PSG). During the prepupal period, silkglands are degenerated via PCD pathway which is triggered by the steroid hormone ecdysone. The pupal differentiation of silkgland starts on the first day of the fifth instar and cell death in ASG is initiated on the third day. Both apoptotic and autophagic morphologies have been observed in the ASG during the larval-pupal metamorphosis, but the connections between them are still unclear. Besides the PCD phenomenon found in ASG, we have also observed autophagic vacuolar formation in MSG during the prepupal period (unpublished data, Cao et al.). These findings indicate that autophagy may play a very important part in the differentiation and degeneration of silkgland, but the molecular mechanism remains unclear. Thus we performed a genome-wide search of autophagy-related genes in B. mori, aiming at remodeling the preliminary autophagy pathway for further systematic investigations.Based on the autophagy pathway in several model organisms and a series of bioinformatics analyses, we have found more than 20 autophagy-related genes from the current database of the silkworm B. mori. Bioinformatics in combination with RT-PCR enable us to remodel a preliminary pathway of autophagy in the silkworm. Amplification and cloning of most autophagy-related genes from the silkgland indicated autophagy is indeed an activated process. Furthermore, the time-course transcriptional profiles of BmATG8 and BmATG12 revealed that both genes are up-regulated along the maturation of the silkgland during the fifth instar. These findings suggest that the autophagy should play an important role in B. mori silkglands.