The crystal structure of (3R)-hydroxyacyl-CoA dehydrogenase of rat peroxisomal multifunctional enzyme type 2 (MFE-2) was solved at 2.38 A resolution. The catalytic entity reveals an alpha/beta short chain alcohol dehydrogenase/reductase (SDR) fold and the conformation of the bound nicotinamide adenine dinucleotide (NAD(+)) found in other SDR enzymes. Of great interest is the separate COOH-terminal domain, which is not seen in other SDR structures. This domain completes the active site cavity of the neighboring monomer and extends dimeric interactions. Peroxisomal diseases that arise because of point mutations in the dehydrogenase-coding region of the MFE-2 gene can be mapped to changes in amino acids involved in NAD(+) binding and protein dimerization.
Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulated during early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoA reductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCR1 and 2 contain open reading frames of 963 bp and 924 bp encoding proteins of 320 and 307 amino acid residues, respectively. Quantatitive RT-PCR analysis showed that both these genes were highly preferentially expressed during the cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2 showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAs were cloned and expressed in yeast haploid ybr159w∆ mutant that was deficient in 3-ketoacyl-CoA reductase activity. Wild-type growth rate was restored in ybr159w∆ cells that expressed either GhKCR1 or 2. Further analysis showed that GhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to the yeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductase activity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest that GhKCR1 and 2 are functional orthologues of ScYbr159p.
Chondroitin sulfate (CS) and dermatan sulfate (DS) are widely distributed on the cell surface and in the extracellular matrix in the form of proteoglycan, where they participate in various biological processes. The diverse functions of CS/DS can be mainly attributed to their high structural variability. However, their structural complexity creates a big challenge for structural and functional studies of CS/DS. CS/DS-degrading enzymes with different specific activities are irreplaceable tools that could be used to solve this problem. Depending on the site of action, CS/DS-degrading enzymes can be classified as glycosidic bond-cleaving enzymes and sulfatases from animals and microorganisms. As discussed in this review, a few of the identified enzymes, particularly those from bacteria, have wildly applied to the basic studies and applications of CS/DS, such as disaccharide composition analysis, the preparation of bioactive oligosaccharides, oligosaccharide sequencing, and potential medical application, but these do not fulfill all of the needs in terms of the structural complexity of CS/DS.
The tubulin beta III (TUBB3) gene encodes a class III member of the beta tubulin protein family that is primarily expressed in neurons and is considered to play a critical role in proper axon guidance and maintenance. This protein is generally used as a specific marker of neurons in the central nervous system. We obtained the full length cDNA sequence of TUBB3 by using the RACE method based on the EST fragment from the brain and spinal cord cDNA library of Gekko japonicus. We further investigated the multi-tissue expression pattern by RT-PCR and identified one transcript of TUBB3 about 1.8 kb in the central nervous system of Gekko japonicus by Northern blotting. The completed cDNA of gecko TUBB3 is 1 790 bp with an open reading frame of 1 350 bp, encoding a 450 amino-acid protein. The recombinant plasmid of pET-32a-TUBB3 was constructed and induced to express His-tagged TUBB3 protein in prokaryotic BL21 cells. The purified TUBB3 protein was then used to immunize rabbits to generate polyclonal antisera. The titer of the antiserum was more than 1:65 536 determined by ELISA. The result of western blotting showed that the TUBB3 antibody could specifically recognize the recombinant TUBB3 protein and endogenous TUBB3 protein. Our findings provide the tools to further understand the TUBB3 gene and investigate the regeneration of the central nervous system in Gekko japonicas.
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