Cofactor specificity

Many redox enzymes used in BFCs require cofactors for catalysis. For example, yeast alcohol dehydrogenase catalyzes the oxidation of ethanol to acetaldehyde, with the concomitant reduction of NAD to NADH. Most commonly, cofactor specificity has been engineered to increase the activity of NADP(H)-dependent enzymes with NAD(H) or to correct a cofactor imbalance in a metabolic process. As the NAD(H) cofactor is more stable and less expensive than NADP(H), this generally improves the economics of a process [42—44]. 

However, these natural cofactors are not ideal for use in bioelectrocatalytic systems. In addition to being expensive, natural cofactors are only moderately stable and have a limited lifetime in enzymatic systems (Table 7.1). The relatively large size of the cofactors can lead to mass transfer limitations in some BFC architectures. Finally, the high ( 1 V) overpotential required to oxidize NAD(P)H at an electrode significantly reduces the efficiency of the reaction to far below that of the theoretical limit. The overpotential can be lowered through the addition of redox mediators to the system, but they also possess relatively poor stability and have a limited lifetime. 

To address these limitations, the use of alternative biomimetic cofactors has been investigated. In this way, a cofactor with the desired size, charge, redox potential, regeneration kinetics, or any other features deemed important for the 

TABLE 7.1 Properties of Cofactors Commonly Used in Biological Fuel Cells 

Cofactor Cost Molecular Weight Standard Redox Potential ( q, V) Comments 

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Lipke H, CW Kearns (1959b) DDT dehydrochlorinase II. Substrate and cofactor specificity. J Biol Chem 234 ... 

Fe2S2] clusters are part of the molybdenum containing hydroxylases. Typically, apart from molybdenum and two EPR-distinct iron-sulfur centres there can be FAD as additional cofactor. In Chlostridium purinolyticum a selenium-dependent purine hydroxylase has been characterized as molybdenum hydroxylase. The EPR of the respective desulfo molybdenum (V) signal indicated that the Mo-ligands should differ from those of the well known mammalian corollary xanthine oxidase.197 For the bacterial molybdenum hydroxylase quinoline oxidoreductase from Pseudomonas putida an expression system was developed in order to be able to construct protein mutants for detailed analysis. EPR was used to control the correct insertion of the cofactors, specifically of the two [Fe2S2] clusters.198... 

However, this glycine-rich segment has other functions in short-chain alcohol dehydrogenases. Tanaka et al. [37] and our 3D modeling [60] indicate that this glycine rich segment has an important role in cofactor specificity and binding of the nicotinamide moiety to 11P-HSD. 

Chen Z, Tsigelny I, Lee WR, Baker ME, Chang SH. Adding a positive charge at residue 46 of Drosophila alcohol dehydrogenase increases cofactor specificity for NADP+. FEBS Lett 356 1994 81-85. 

JH Hurley, RD Chen, AM Dean. Determinants of cofactor specificity in isocitrate dehydrogenase structure of an engineered NADP —> NAD specificity-reversal mutant. Biochemistry 35 5670-5678, 1996. 

JH Hurley, AM Dean. Structure of 3-isopropylmalate dehydrogenase in complex with NAD+ ligand-induced loop closing and mechanism for cofactor specificity. Structure 2 1007-1016, 1994. 

During natural evolution, a broad variety of enzymes has been developed, which are classified according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). Thus, for each type of characterized enzyme an EC (Enzyme Commission) number has been provided (see http // www.expasy.ch/enzyme/). For instance, all hydrolases have EC number 3 and further subdivisions are provided by three additional digits, e.g. all lipases (official name triacylglycerol lipases) have the EC number 3.1.1.3 and are thus distinguished from esterases (official name carboxyl esterases) having the EC number 3.1.1.1. This classification is based on the substrate (and cofactor) specificity of an enzyme only, however often very similar amino acid sequences and also related three-dimensional structures can be observed. 

These chelating resins have found most of their use in metal ion recovery processes in the chemical and waste recovery industries. They may find use in fermentation applications where the cultured organism requires the use of metal ion cofactors. Specific ion exchange resins have also been used in laboratory applications that may find eventual use in biotechnology product recovery applications. 

As all mutation sites chosen in this study are limited to the (lap fingerprint motif, the strategy applied is applicable to other NAD+- and NADP+-dependent dehydrogenases. Indeed, a systematic replacement of amino acid residues in the Pap fingerprint motif in the NAD+-dependent dihydrolipoamide dehydrogenase from E. coli converted its cofactor specificity from NAD+ to NADP+14IL A similar strategy... 

Enzyme and source Number of proteins Subunit structure Cofactor specificity Native molecular mass x 1000 Da Mole of flavin/ mole of protein Optimum pH Reference... 

E. coli or other known proteins. This approach has been used extensively in our laboratory for the genes of fatty acid synthesis, with isoforms from pathogenic bacteria being isolated to assess their unique biochemical characteristics and for use in drug screening programs. Subtle differences in cofactor specificity can be detected that are not obvious from the primary sequences. 

With regard to cofactor specificity, it is interesting that saFabI is NADPH-dependent, whereas the FabI homologs from E. coli (ecFabI), Bacillus subtilis, Haemophilus influenzae, and M. tuberculosis are all NADH-dependent enoyl-ACP reductases. According to the ecFabI—NAD crystal structure, Q40 is very close (2.8 A) to the 2 -hydroxyl group of the NAD adenosine moiety (Figure 20). Although the crystal... 

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