The Structural And Functional Researches Of Yeast Mitochondria3-Oxoacyl-ACP Reductase

Fatty acid is a kind of very important hydrophobic molecule which plays a variety of roles in the cell. Fatty acid synthetase (FAS), a complex enzyme system, is responsible for de novo fatty acid synthesis from acetyl CoA or malonyl CoA in nature. Two main types of this enzyme system are found in a variety of species. The type Ⅰ FAS system is distributed in vertebrates, yeast cytoplasmic matrix and some bacteria. It is a single, large multifunctional polypeptide that catalyzes reactions involved in the elongation of the fatty acid chain. For many bacteria and plants, the type Ⅱ FAS system plays a major role in this process as a series of separate enzymes. Subsequent studies on FAS in organelles have revealed that there exists a mitochondrial type Ⅱ FAS in Saccharomyces cerevisiae. Mitochondrial FAS generates octanoyl-ACP used for the synthesis of lipoyl moieties and is also linked to RNA processing in both yeast and mammals. Yeast contains six genes encoding components of mitochondrial type Ⅱ FAS:ACPI, CEM1, MCT1, HTD2, ETR1and OARI. The OARI gene is responsible for encoding mitochondrial beta-ketoacyl-ACP reductase (YmtOAR1) and recent studies suggest that deletion of OARI leads to a respiratory deficient phenotype and causes growth inhibition.Sequence alignments have shown that YmtOAR1belongs to a short-chain alcohol dehydrogenase (SDR) family and is20%-30%identical to homologous enzymes. Unlike other SDR family members. YmtOAR1forms a homodimer in solution. The overall structure of YmtOAR1does not show a canonical Rossmann fold due to the inserted sequence and the deletion of C-terminal. During the enzymatic reaction, with a hydride transfer from coenzyme NADPH followed by proton donation from YmtOAR1, the substrate was reduced. Here, we present the crystal structure of the YmtOAR1alone in apo-form at2.60A and complexed with NADPH at2.10A resolution. The enzyme generates significant conformational changes upon NADPH binding to the active site. Moreover, two different cofactor-binding patterns are observed from two forms of complex crystals. The two forms of NADPH bound enzyme may mimic the dynamic process of cofactor recognition. In addition, we found that the mutation of Argl4to alanine causes a loss of enzymatic activity and greatly decreases cofactor affinity. Structural and biochemical studies enable us to further understand the3-oxoacyl-(acyl-carrier-protein) reductases (OARs) of FAS and also provide some implications for cofactor recognition.Eukaryotic membrane system is very sophisticated, not only to separate the place where a variety of biochemical reactions involved in, but also to exchange the substances between the various organelles-vesicle trafficking. The vesicles envelop cargo molecules, with the assistance of some special proteins, for example, SNARE or Sorting Nexins family, and take part in membrane fusion or budding, thereby completing the sorting and transport of cargos.Mvp1protein is responsible for the sorting pathway between Golgi apparatus and late lysosome in Saccharomyces cerevisiae. As a unique gene, MVP1, that exhibits genetic interaction with VPS1and is itself required for vacuolar protein sorting has been isolated. Overproduction of Mvplp will suppress several dominant alleles of VPS1. The sorting mechanism of Mvpl involved in the vesicle transport process has not yet been elucidated. In this study, We crystallized the fragment of Mvp1(⊿N117-Mvp1), which attempts to resolve the crystal structure. Unfortunately, the crystals showed weak diffraction and further optimization work is still ongoing.