Because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV) is a useful reagent for cleaving genetically engineered fusion proteins. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. The rate of autoinactivation is proportional to the concentration of TEV protease, implying a bimolecular reaction mechanism. Yet, a catalytically active protease was unable to convert a catalytically inactive protease into the truncated form. Adding increasing concentrations of the catalytically inactive protease to a fixed amount of the wild-type enzyme accelerated its rate of autoinactivation. Taken together, these results suggest that autoinactivation of TEV protease may be an intramolecular reaction that is facilitated by an allosteric interaction between protease molecules. In an effort to create a more stable protease, we made amino acid substitutions in the P2 and P1' positions of the internal cleavage site and assessed their impact on the enzyme's stability and catalytic activity. One of the P1' mutants, S219V, was not only far more stable than the wild-type protease (approximately 100-fold), but also a more efficient catalyst.

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In the presence of carbon monoxide, the photosynthetic bacterium Rhodospirillum rubrum induces expression of proteins which allow the organism to metabolize carbon monoxide in the net reaction CO ؉ H 2 O 3 CO 2 ؉ H 2 . These proteins include the enzymes carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. In this paper, we present the complete amino acid sequence for the large subunit of this hydrogenase and describe the properties of the crude enzyme in relation to other known hydrogenases. The amino acid sequence deduced from the CO-induced hydrogenase large-subunit gene (cooH) shows significant similarity to large subunits of other Ni-Fe hydrogenases. The closest similarity is with HycE (58% similarity and 37% identity) from Escherichia coli, which is the large subunit of an Ni-Fe hydrogenase (isoenzyme 3). The properties of the CO-induced hydrogenase are unique. It is exceptionally resistant to inhibition by carbon monoxide. It also exhibits a very high ratio of H 2 evolution to H 2 uptake activity compared with other known hydrogenases. The CO-induced hydrogenase is tightly membrane bound, and its inhibition by nonionic detergents is described. Finally, the presence of nickel in the hydrogenase is addressed. Analysis of wild-type R. rubrum grown on nickel-depleted medium indicates a requirement for nickel for hydrogenase activity. However, analysis of strain UR294 (cooC insertion mutant defective in nickel insertion into CODH) shows that independent nickel insertion mechanisms are utilized by hydrogenase and CODH. CooH lacks the C-terminal peptide that is found in other Ni-Fe hydrogenases; in other systems, this peptide is cleaved during Ni processing.The purple nonsulfur bacterium Rhodospirillum rubrum contains a CO-induced hydrogenase that is a component of the CO-oxidizing system of this organism (12). This system allows R. rubrum to grow in a CO-dependent manner in the dark (25). The key proteins of this membrane-bound enzyme complex are induced when the organism is exposed to carbon monoxide (12). Carbon monoxide dehydrogenase (CODH), a 62-kDa peripheral membrane protein, carries out the primary oxidation of carbon monoxide to carbon dioxide (13) and passes the resulting reducing equivalents through a 22-kDa ferredoxin-like subunit. This ferredoxin passes the electrons to uncharacterized electron carriers and then eventually to a tightly membranebound hydrogenase, where protons are reduced to dihydrogen (18).R. rubrum contains several other known hydrogenases in addition to the one induced by CO. These are involved with growth under CO 2 /H 2 (20), fermentative growth on substrates like pyruvate (42), and recycling of H 2 produced by nitrogenase under nitrogen-fixing conditions (26).The genes encoding R. rubrum CODH (cooS), the ferredoxin-like CODH small subunit (cooF), and several genes involved in nickel insertion into CODH (cooCTJ) have been sequenced and characterized (24, 25a). The cooA gene, located 3Ј of cooFSCTJ, has also been identified recently, and its product is required for ...

In the photosynthetic bacterium Rhodospirillum rubrum, the presence of carbon monoxide (CO) induces expression of several proteins. These include carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. Together these enzymes catalyze the following conversion: CO ؉ H 2 O 3 CO 2 ؉ H 2 . This system enables R. rubrum to grow in the dark on CO as the sole energy source. Expression of this system has been shown previously to be regulated at the transcriptional level by CO. We have now identified the remainder of the CO-regulated genes encoded in a contiguous region of the R. rubrum genome. These genes, cooMKLXU, apparently encode proteins related to the function of the CO-induced hydrogenase. As seen before with the gene for the large subunit of the CO-induced hydrogenase (cooH), most of the proteins predicted by these additional genes show significant sequence similarity to subunits of Escherichia coli hydrogenase 3. In addition, all of the newly identified coo gene products show similarity to subunits of NADH-quinone oxidoreductase (energyconserving NADH dehydrogenase I) from various eukaryotic and prokaryotic organisms. We have found that dicyclohexylcarbodiimide, an inhibitor of mitochondrial NADH dehydrogenase I (also called complex I), inhibits the CO-induced hydrogenase as well. We also show that expression of the cooMKLXUH operon is regulated by CO and the transcriptional activator CooA in a manner similar to that of the cooFSCTJ operon that encodes the subunits of CODH and related proteins.In the presence of carbon monoxide (CO), Rhodospirillum rubrum induces synthesis of a CO-oxidizing system. This system catalyzes the net reaction CO ϩ H 2 O 3 CO 2 ϩ H 2 . The reaction is carried out by two enzymes: CO dehydrogenase (CODH) and a CO-tolerant hydrogenase. CODH is a wellcharacterized nickel-iron enzyme (10) that carries out the oxidation of CO to CO 2 , producing two reducing equivalents. Hydrogenase then consumes these reducing equivalents by the reduction of two protons to H 2 (9, 17). Intermediate electron carriers (including the ferredoxin-like small subunit of CODH [17]) may also be involved in the reaction.The hydrogenase is tightly membrane bound and has yet to be purified, although the sequence of its large subunit (CooH) and several of its properties have been described previously (22). A wide variety of hydrogenases from other organisms, however, have been purified and characterized. These hydrogenases have been found to fall into three distinct categories: Fe-only, Ni-Fe, and Ni-Fe-Se enzymes (for reviews, see references 1, 4, and 56). The CO-induced hydrogenase from R. rubrum apparently belongs to the Ni-Fe class, on the basis of its protein sequence and the requirement of Ni for activity (22).There seem to be two CO-regulated transcripts in R. rubrum. The first, cooFSCTJ, encodes CODH and related proteins (27,29,43) and has been shown to be regulated by the product of the cooA gene (43). CooA appears to bind to a specific region of DNA upstream of the cooF promoter in a CO-dependent m...

Proteins are commonly fused to Escherichia coli maltose-binding protein (MBP) to enhance their yield and facilitate their purification. In addition, the stability and solubility of a passenger protein can often be improved by fusing it to MBP. In a previous comparison with two other highly soluble fusion partners, MBP was decidedly superior at promoting the solubility of a range of aggregation-prone proteins. To explain this observation, we proposed that MBP could function as a general molecular chaperone in the context of a fusion protein by binding to aggregation-prone folding intermediates of passenger proteins and preventing their self-association. The ligand-binding cleft in MBP was considered a likely site for peptide binding because of its hydrophobic nature. We tested this hypothesis by systematically replacing hydrophobic amino acid side chains in and around the cleft with glutamic acid. None of these mutations affected the yield or solubility of MBP in its unfused state. Each MBP was then tested for its ability to promote solubility when fused to three passenger proteins: green fluorescent protein, p16, and E6. Mutations within the maltosebinding cleft (W62E, A63E, Y155E, W230E, and W340E) had little or no effect on the solubility of the fusion proteins. In contrast, three mutations near one end of the cleft (W232E, Y242E, and I317E) dramatically reduced the solubility of the same fusion proteins. The mutations with the most profound effect on solubility were shown to reduce the global stability of MBP.