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We have expressed the DNA of the fusion of CS1 to MDH1 in Escherichia coli gltA-. The fusion protein (CS1/MDH1) is the C-terminus of CS1 linked in-frame to the N-terminus of MDH1 with a short linker of glycyl-seryl-glycyl. The fusion protein produced was isolated and purified. Gel filtration studies indicated that CS1/MDH1 had a M(r) of approximately 170,000. Western blotting analysis with SDS gel indicated a M(r) of approximately 90,000-95,000 (theoretical M(r) = 87,000). This is the expected M(r) for the fusion protein subunit. The kinetics of CS1 and MDH1 activities of the fusion protein were compared to those of the free enzymes. In addition, the effect of AAT reaction, as a competitor for the intermediate OAA of the coupled MDH-CS reaction, was examined. It was observed that AAT was a less effective competitor for OAA when the CS1/MDH1 fusion protein is used than when the separate enzymes are employed. In addition, the transient time for the coupled reaction sequence was less for the fusion protein than for the free enzymes.

The tendency of bacteria to assemble at oil-water interfaces can be utilized to create microbial recognition sites on the surface of polymer beads. In this work, two different groups of bacteria were first treated with acryloyl-functionalized chitosan and then used to stabilize an oil-in-water emulsion composed of cross-linking monomers that were dispersed in aqueous buffer. Polymerization of the oil phase followed by removal of the bacterial template resulted in well-defined polymer beads bearing bacterial imprints. Chemical passivation of chitosan and cell displacement assays indicate that the bacterial recognition on the polymer beads was dependent on the nature of the pre-polymer and the target bacteria. The functional materials for microbial recognition show great potential for constructing cell-cell communication networks, biosensors, and new platforms for testing antibiotic drugs.

L-lactate dehydrogenase (LDH) from Bacillus stearothermophilus is a redox enzyme which has a strong preference for NADH over NADPH as coenzyme. To exclude NADPH from the coenzyme-binding pocket, LDH contains a conserved aspartate residue at position 52. However, this residue is probably not solely responsible for the NADH specificity. In this report we examine the possibilities of altering the coenzyme specificity of LDH by introducing a range of different point mutations in the coenzyme-binding domain. Furthermore, after choosing the mutant with the highest selectivity for NADPH, we also investigated the possibility of further altering the coenzyme specificity by adding an organic solvent to the reaction mixture. The LDH mutant, I51K:D52S, exhibited a 56-fold increased specificity to NADPH over the wild-type LDH in a reaction mixture containing 15% methanol. Furthermore, the NADPH turnover number of this mutant was increased almost fourfold as compared with wild-type LDH. To explain the altered coenzyme specificity exhibited by the D52SI51K double mutant, molecular dynamics simulations were performed.

Respiratory proteins such as myoglobin and hemoglobin can, under oxidative conditions, form ferryl heme iron and proteinbased free radicals. Ferryl myoglobin can safely be returned to the ferric oxidation state by electron donation from exogenous reductants via a mechanism that involves two distinct pathways. In addition to direct transfer between the electron donor and ferryl heme edge, there is a second pathway that involves "through-protein" electron transfer via a tyrosine residue (tyrosine 103, sperm whale myoglobin). Here we show that the heterogeneous subunits of human hemoglobin, the ␣ and ␤ chains, display significantly different kinetics for ferryl reduction by exogenous reductants. By using selected hemoglobin mutants, we show that the ␣ chain possesses two electron transfer pathways, similar to myoglobin. Furthermore, tyrosine 42 is shown to be a critical component of the high affinity, through-protein electron transfer pathway. We also show that the ␤ chain of hemoglobin, lacking the homologous tyrosine, does not possess this through-protein electron transfer pathway. However, such a pathway can be engineered into the protein by mutation of a specific phenylalanine residue to a tyrosine. High affinity through-protein electron transfer pathways, whether native or engineered, enhance the kinetics of ferryl removal by reductants, particularly at low reductant concentrations. Ferryl iron has been suggested to be a major cause of the oxidative toxicity of hemoglobin-based blood substitutes. Engineering hemoglobin with enhanced rates of ferryl removal, as we show here, is therefore likely to result in molecules better suited for in vivo oxygen delivery.