Crystal Structures of Arabidopsis thaliana GDP-D-Mannose Pyrophosphorylase VITAMIN C DEFECTIVE 1.

Plant GDP-D-mannose pyrophosphorylase (GMPase) catalyzes a committed step in ascorbic acid biosynthesis pathway. Arabidopsis thaliana VTC1 is the first genetically characterized plant GMPase and has unique properties when compared with bacterial and animal homologs. Here we present the crystal structures of VTC1 in the unliganded and product-bound states at resolutions of 2.8 and 3.0 Å, respectively. VTC1 dimerizes in a same way like other known GMPases, but dodecamerizes in a previously unobserved arrangement. The interactions to GDP-D-mannose and inorganic pyrophosphate are revealed by the product-bound VTC1 structure. An in vitro GMPase activity assay confirms the regulatory role of the C-terminal left-handed ß-helix domain, and structural analyses suggest the models of VTC1 hetero-complex with its interacting proteins. The structural information advances our insights into the different mechanisms involved in VTC1 regulation.

Crystal structures of cyanobacterial light-dependent protochlorophyllide oxidoreductase.

The reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) is the penultimate step of chlorophyll biosynthesis. In oxygenic photosynthetic bacteria, algae, and plants, this reaction can be catalyzed by the light-dependent Pchlide oxidoreductase (LPOR), a member of the short-chain dehydrogenase superfamily sharing a conserved Rossmann fold for NAD(P)H binding and the catalytic activity. Whereas modeling and simulation approaches have been used to study the catalytic mechanism of this light-driven reaction, key details of the LPOR structure remain unclear. We determined the crystal structures of LPOR from two cyanobacteria, Synechocystis sp. PCC 6803 and Thermosynechococcus elongatus Structural analysis defines the LPOR core fold, outlines the LPOR-NADPH interaction network, identifies the residues forming the substrate cavity and the proton-relay path, and reveals the role of the LPOR-specific loop. These findings provide a basis for understanding the structure-function relationships of the light-driven Pchlide reduction.

AZFa Microdeletions: Occurrence in Chinese Infertile Men and Novel Deletions Revealed by Semiconductor Sequencing.

OBJECTIVE: To evaluate the frequency of azoospermia factor (AZFa) microdeletions among infertile men and establish a new high-throughput sequencing method to detect novel deletion types. MATERIALS AND METHODS: A total of 3731 infertile men were included. Karyotype analysis was performed using G-band staining of peripheral blood lymphocytes. Polymerase chain reaction (PCR) amplification using specific sequence-tagged sites (STS) was performed to screen for AZF region microdeletions of the Y chromosome. A novel semiconductor sequencing method was established to detect high-resolution AZFa microdeletions. RESULTS: Of 3731 infertile men, 341 (9.14%) had microdeletions in AZFa, AZFb, or AZFc. Thirteen of these (3.81%) had a deletion in the AZFa region (mean age: 27.3 ± 4 years, range: 22-34), which included 12 subjects with a normal karyotype (46, XY) and 1 with Klinefelter syndrome (47, XXY). Four of 10 subjects with complete AZFa microdeletions (sY86 and sY84 loss) underwent semiconductor sequencing. They all had DNA sequence deletions from nt 14469266 to 15195932, whereas their fathers had no deletions. One subject with partial AZFa microdeletion (sY86 loss) and his father underwent semiconductor sequencing and STS-PCR analysis. The same deletion (sY86 loss with DNA sequence deletion from nt 14469266 to 14607672) was identified in both subjects. Forty sperm donators and 50 infertile men showed no AZFa microdeletions by either method. CONCLUSION: AZFa deletions are present at a low frequency in men with azoospermia or oligozoospermia. Novel sequencing methods can be used for these patients to reveal high-resolution AZFa microdeletions.