"The amplified genes of both 10093b and 10093f were gel purified and cloned into pSGX3 (BC) vector designed to express the protein of interest with a C-terminal hexa-histidine tag to facilitate easy and high yield purification. Protein expression/purification utilized previously published protocols . For 10093b a yield of 22 mg was obtained from 3L culture, whereas for 10093f the yield was 91 mg from 2L culture. "
[Show abstract][Hide abstract] ABSTRACT: Pfam is a comprehensive collection of protein domains and families, with a range of well-established information including genome annotation. Pfam has two large series of functionally uncharacterized families, known as Domains of Unknown Function (DUFs) and Uncharacterized Protein Families (UPFs).
Crystal structures of two proteins from Deinococcus radiodurans and Streptomyces coelicolor belonging to Pfam protein family DUF178 (ID: PF02621) have been determined using Selenium-Single-wavelength Anomalous Dispersion (Se-SAD). Based on the structure, we have identified the putative function for this family of protein.
Unexpectedly, we found that DUF178 Pfam is remarkably similar to Pfam family DUF191 suggesting that the sequence-based classification alone may not be sufficient to classify proteins into Pfam families.
[Show abstract][Hide abstract] ABSTRACT: periplasmic glycerophosphodiester phosphodiesterase, GlpQ. UgpQ has broad substrate specificity toward various glycerophosphodiesters, producing sn-glycerol-3-phosphate and the corresponding alcohols. UgpQ accumulates under conditions of phosphate starvation, suggesting that it allows the utilization of glycerophos- phodiesters as a source of phosphate. These results clarify how E. coli utilizes glycerophosphodiesters using two homologous enzymes, UgpQ and GlpQ. Glycerophosphodiesters are enzymatically produced by phospholipases A1 and A2 from membrane phospholipids (10, 25). There are several glycerophosphodiesters, based on the alcohol moiety: glycerophosphocholine (GPC), glycerophos- phoethanolamine (GPE), glycerophosphoinositol (GPI), glyc- erophosphoserine (GPS), glycerophosphoglycerol (GPG), and so on. Glycerophosphodiesters are further degraded by glyc- erophosphodiester phosphodiesterase (EC 184.108.40.206), produc- ing the corresponding alcohols and sn-glycerol-3-phosphate (G3P), which is an essential precursor for de novo synthesis of glycerophospholipids. In the metabolic pathway of Escherichia coli, glycerophosphodiesters are thought to be utilized by two distinct systems. One is the Glp system, and the other is the Ugp system (26). In each system, related proteins are encoded by one or more operons. They contain genes that code for transporter proteins and enzymes. The glpTQ operon simultaneously regulates the transcrip- tion of related genes (13). GlpT, an ABC transporter, actively transports G3P from the periplasm into the cytosol through the plasma membrane. glpQ codes for a periplasmic glycerophos- phodiester phosphodiesterase. GlpQ processes periplasmic glycerophosphodiesters into G3P and corresponding alcohols (14). The other glycerophosphodiester-utilizing system is the Ugp system. The ugp operon is constituted of ugpB, ugpA, ugpE, ugpC, and ugpQ (4, 19). UgpB specifically binds to glycero- phosphodiesters and delivers them to the membrane trans- porter. UgpA, UgpE, and UgpC constitute the transporter for glycerophosphodiesters. UgpQ, a 27-kDa protein, is a cytosolic glycerophosphodiester phosphodiesterase and is homologous to GlpQ.
[Show abstract][Hide abstract] ABSTRACT: The structure of the glycerophosphodiesterase (GDPD) from Enterobacter aerogenes, GpdQ, has been solved by SAD phasing from the active site metal ions. Structural analysis indicates that GpdQ belongs to the alpha/beta sandwich metallo-phosphoesterase family, rather than the (alpha/beta)(8) barrel GDPD family, suggesting that GpdQ is a structurally novel GDPD. Hexameric GpdQ is generated by interactions between three dimers. The dimers are formed through domain swapping, stabilised by an inter-chain disulfide bond, and beta-sheet extension. The active site contains a binuclear metal centre, with a fully occupied alpha-metal ion site, and partially occupied beta-metal ion site, as revealed by anomalous scattering analysis. Using a combination of TLS refinement and normal mode analysis, the dynamic movement of GpdQ was investigated. This analysis suggests that the hexameric quaternary structure stabilises the base of the dimer, which promotes "breathing" of the active site cleft. Comparison with other metallo-phosphodiesterases shows that although the central, catalytic, domain is highly conserved, many of these enzymes possess structurally unrelated secondary domains located at the entrance of the active site. We suggest that this could be a common structural feature of metallo-phosphodiesterases that constrains substrate specificity, preventing non-specific phosphodiester hydrolysis.
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