Recombinant formate dehydrogenase from the acetogen Clostridium carboxidivorans strain P7 T , expressed in Escherichia coli, shows particular activity towards NADH-dependent carbon dioxide reduction to formate due to the relative binding affinities of the substrates and products. The enzyme retains activity over 2 days at 4°C under oxic conditions. F ormate dehydrogenases (FDHs) catalyze the interconversion of CO 2 and formic acid through an oxidoreductive process ( Fig. 1) (1). Consequently, when catalyzing CO 2 reduction, they are of interest for the sequestration of CO 2 and for the production of formic acid as a stabilized form of hydrogen fuel and as a source of commodity chemicals. In many bacteria and eukaryotes, FDHs catalyze the final step of catabolic processes in which formate is oxidized to CO 2 (2). The ability of certain members of this class, such as FDH from Candida boidinii in particular, to efficiently regenerate NADH in conjunction with formate oxidation has been a research focus (3).Acetogens are known to possess a number of pathways distinct from those found in the other species. FDHs present in acetogens are known to take part in a carbon fixation metabolic pathway producing acetate (the Eastern branch of the Wood-Ljungdahl pathway), in which the first step involves reduction of CO 2 to formate (4). Several FDHs are known to catalyze CO 2 reduction under appropriate conditions (5-9). Those enzymes from acetogenic and related anaerobes, such as Moorella thermoacetica and Clostridium pasteurianum, are better than other FDHs as reduction catalysts but also show similar catalytic efficiency toward formate oxidation and are considered highly oxygen labile, requiring anaerobic expression and purification as well as anoxic assay conditions (10, 11). Clostridium carboxidivorans strain P7 T (equivalent to ATCC BAA-624 T and DSM 15243 T ) was isolated from the sediment of an agricultural settling lagoon after enrichment with CO as the substrate and is an obligate anaerobe that can grow autotrophically with H 2 and CO 2 or CO (fixing carbon via the Wood-Ljungdahl pathway) (12, 13). Therefore, when the gene of a selenocysteine-containing formate dehydrogenase H (FDH H ) from the acetogen Clostridium carboxidivorans strain P7T was first identified, it was suggested that FDH H would catalyze the conversion of CO 2 to formate (14, 15). Here we report the first production of FDH H and its catalytic preference for CO 2 reduction, as well as its tolerance for oxic conditions. Cloning, expression, and purification of FDHs. The overexpression and purification of recombinant FDH H from the Clostridium carboxidivorans strain P7 T (FDH H _CloCa) was carried out, along with that of NAD ϩ -dependent recombinant FDH from Candida boidinii (FDH_CanBo), in order to compare expression and activity of formate dehydrogenases that take part in distinct metabolic pathways. The DNA sequences for the FDH H _CloCa (UniProt E2IQB0) and FDH_CanBo (UniProt O13437) genes were codon optimized for expression in Escherichia coli (com...
E. coli lysate efficiently catalyzes acetyl phosphate-driven ATP regeneration in several important biotechnological applications. The utility of this ATP recycling strategy in enzyme-catalyzed chemical synthesis is illustrated through the conversion of uridine to UMP by the lysate from recombinant overexpression of uridine kinase with the E. coli. The UMP is further transformed into UTP through sequential phosphorylations by kinases naturally present in the lysate, in high yield. Cytidine and 5-fluorouridine also give the corresponding NMPs and NTPs with this system. Cell-free protein expression with a processed extract of lysate also proceeds readily when, instead of adding the required NTPs, all four are produced in situ from the NMPs, using acetyl phosphate and relying on endogenous kinase activity. Similarly, dNMPs can be used to produce the dNTPs necessary for DNA synthesis in PCR. These cheap alternative protocols showcase the potential of acetyl phosphate and ATP recycling with readily available cell lysate.
The recently reported F 420 H 2 -dependent reductases (FDRs) catalyse the reduction of aflatoxins and coumarin via hydrogenation of the a,b-unsaturated moiety. We report that three FDRs (MSMEG_2027, MSMEG_6848 and MSMEG_3356) from Mycobacterium smegmatis also exhibit a different catalytic function towards some aflatoxins through the use of a different cofactor. When F 420 was replaced by FMN in these three enzymes, the aflatoxins AFG1 and AFG2 were oxidised via dehydrogenation, producing the reduced cofactor (FMNH 2 ) and an unstable aflatoxin derivative that hydrolyses to an enol with three distinct structural isomers. Both the oxidation and reduction reactions are discussed in detail. This is the first example of an enzyme showing promiscuity for its cofactor leading to divergence of function against the same substrate.
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