| Oxaloacetate decarboxylases (OAD, EC4.1.1.3) catalyze the decarboxylation of oxaloacetate to pyruvate and CO2. This family generally includes two types of enzymes: one is membrane bound oxaloacetate decarboxylase sodium pump subfamily, and the other is cytosolic soluble oxaloacetate decarboxylase subfamily.The gene encoding for the cytosolic oxaloacetate decarboxylase in Corynebacterium glutamicum was determined recently. The sequence alignment showed that Cg1458has no similarity with all known oxaloacetate decarboxylase, but has reasonable high similarity with the members from fumarylacetoacetate hydrolase (FAH) family. FAH is a metabolic enzyme catalyzing the final step of tyrosine and phenylalanine catabolism, the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate to yield fumarate and acetoacetate, which then joins the citric acid cycle. Some members from FAH family have been extensively studied with divergent functions, but none of them has been reported with oxaloacetate decarboxylase activity previously.The membrane bounded oxaloacetate decarboxylase belongs to the family of Na+translocating decarboxylase that couple the exergonic chemical reaction to an active Na+transport to the outside of the cell across the membrane. The Na+gradient thus established across the cell membrane can be used to drive solute uptake, flagellar motion or the synthesis of ATP by an F-type ATP synthase. Although the crystal structure of the transcarboxylase domain of the alpha subunit from V. cholerae was determined, but the mechanism of the sodium coupled oxaloacetate decarboxylase is still uncovered. To unveal the mechanism of Oad complex, high quality protein needs to be used for getting high quality crystals. The extensive studies for getting high yield and homogenous complex are necessary for futural structure determination.To reveal the catalytic mechanism of a novel oxaloacetate decarboxylase Cg1458, we cloned, expressed and crystallized the novel oxaloacetate decarboxylase (Cg1458) from C. glutamicum which is a FAH family member from the sequence analysis. The purified enzyme was characterized. The result showed that the optimum pH is Tris-HCl pH8.0. Previous studies showed that the soluble oxaloacetate decarboxylases are divalent metal ion dependent. To determine the importance of metal ions in Cg1458, we assayed the metal depletion and metal reconstituted Cg1458. The result showed that the depletion of the metal ion from Cg1458with EDTA totally abolished the decarboxylase activity, the reconstituted enzymes have even higher activity than the native enzyme when reconstituted with Mn2+, Mg2+, Co2+, Ca2+, while for others (Cu2+, Ni2+and Zn2+), the reconstituted enzymes have much low activity compared with the native enzyme, this result indicated that Cu2+, Ni2+and Zn2+are not suitable for the enzyme activity. Since the reconstituted enzymes with some ions have higher activity than native protein, we measured the activity of enzyme with different additional metal ions. The results showed that the activity of the native Cg1458is increased by the addition of Mn2+, Mg2+, Co2+, Ca2+, Cu2+, Ni2+, whereas Zn2+inhibits the activity of Cg1458. The biochemical characterization showed that oxalate is a potent inhibitor of Cg1458;5mM oxalate could totally inhibits the activity of Cg1458. To elucidate the catalytic mechanism of Cg1458, Cg1458was purified by IMAC and gel filtration, and then crystallized using vapor diffusion method. Full datasets were collected for native Cg1458and oxlate co-crystallized Cg1458, open (the native structure) and close (the oxalate bound structure) conformation crystal structures of Cg1458were determined to1.9A and2.0A resolution, respectively. For the native Cg1458structure, there is a missing loop fragment in the structure which is near the presumably catalytic site of the protein, while in the oxalate bound structure, this fragment is clearly resolved. This fragment serves as a lid which covers the substrate accessing channel and may stop the substrate to enter the active site during each reaction cycle. After the release of product, the conformation of the protein turns to open conformation, and the lid domain becomes flexible. With the structure based sequence analysis of Cg1458, we found some potential active sites. To test the importance of these residues on the OAA decarboxylase activity, we mutated the His73, Glu76, Phe78located in the lid domain, metal ion coordinated residues Glu116, Glu118, Asp147and the residues Arg151, Gln154, Lys164which interacted with the metal coordinated residues to alanine or glycine respectively. The result showed that the mutation of His73, Phe78, Glu116, Glu118,Arg151, Gln154and Lys164abolished the oxaloacetate decarboxylase function, while the mutation of Glu76made the protein lose about65%activity. With detailed structural comparison and sequence alignment, we proposed a common catalytic mechanism for the members of FAH family which catalyze the cleavage of C-C bond of the substrate.To unveil the catalytic mechanism of Oad complex, homogenous and large amount Oad protein is needed. Previous studies showed that the purified Oad complex was not stable. To obtain high amount and homogenous Oad complex for crystallization, we cloned and expressed the recently identified oad genes from Staphylothermus marinus F1in E. coli, we also expressed beta subunit alone and beta subunit fused with other subunits in E. coli, or expressed the constructs with his-tag inserted in different positions respectively in the loop regions of beta subunit according to the topology. Our results showed that the oad genes from S. marinus F1were not expressed in E. coli, the beta subunit and the fused beta subunit were not expressed in E. coli, for the his-tag insertion constructs, only the construct with his-tag inserted at position256could functionally expressed.Proteases play indispensable functions in all living cells such as food digestion, blood clotting, hemocytolysis, inflammation, blood pressure regulation, apoptosis, cancer metastasis and protein activation. Serine proteases comprise nearly one-third of all known proteases identified to date and play crucial roles in a wide variety of cellular as well as extracellular functions including the process of blood clotting, protein digestion, cell signaling, inflammation and protein processing.TTHA1296is a putative serine protease which uses Ser/Lys dyads as catalytic residue from Thermus thermophilus HB8. To know the hydrolytic character, function and probably catalytic mechanism of TTHA1296, we found a substrate Oad525which could be specifically digested by TTHA1296, and characterized TTHA1296by the substrate. To identify the cleavage site of TTHA1296, we choose the Oad525and Oad454which was expressed as the same way as the substrate for TTHA1296, the result showed that TTHA1296could hydrolyze Oad525while Oad454was not a suitable substrate for TTHA1296. So we suppose that TTHA1296is a C teminal protease. The purified TTHA1296was characterized. The results showed that the optimum pH is6.0and the optimum temperature is55℃.TTHA1296is not inhibited by the classical serine protease inhibitors. We crystallized the TTHA1296S295A mutation and got crystals that could diffract to3A. We also co-crystallized TTHA1296S295A and Oad525, plate like crystals was obtained. The best crystal diffracted to2.45A. Structure determination was not successful with molecular replacement using the structures of a homologue as template, the reason for that may be due to the low identity of TTHA1296with its homologue (less than30%identity). The structure will be determined by using experimental phasing method in the future. |
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