Amino acid reductases

Table 12.1 summarizes the selenoenzymes isolated from Clostridia, which fall into three major categories. The first group is the amino acid reductases, such as GR, sarcosine reductase (SR), betaine reductase (BR), and proline reductase (PR). The second class of selenoenzymes includes FDH. Although a number of clostridial species are suspected to contain a... 

The NAD- and NADP-dependent dehydrogenases catalyze at least six different types of reactions simple hydride transfer, deamination of an amino acid to form an a-keto acid, oxidation of /3-hydroxy acids followed by decarboxylation of the /3-keto acid intermediate, oxidation of aldehydes, reduction of isolated double bonds, and the oxidation of carbon-nitrogen bonds (as with dihydrofolate reductase). 

The structure of the UQ-cyt c reductase, also known as the cytochrome bc complex, has been determined by Johann Deisenhofer and his colleagues. (Deisenhofer was a co-recipient of the Nobel Prize in Chemistry for his work on the structure of a photosynthetic reaction center [see Chapter 22]). The complex is a dimer, with each monomer consisting of 11 protein subunits and 2165 amino acid residues (monomer mass, 248 kD). The dimeric structure is pear-shaped and consists of a large domain that extends 75 A into the mito-... 

Overproduction of the chromosomal genes for the dihydrofolate reductase (DHFR) and the dihydroptero-ate synthase (DHPS) leads to a decreased susceptibility to trimethoprim and sulfamethoxazol, respectively. This is thought to be the effect of titrating out the antibiotics. However, clinically significant resistance is always associated with amino acid changes within the target enzymes leading to a decreased affinity of the antibiotics. 

It is not clear why some organisms have two 14-3-3 isoforms while others have up to 12. Binding 14-3-3 inhibits the plant enzyme nitrate reductase and there appears to be no selectivity between plant 14-3-3 isoforms in fact yeast and human isoforms appear to work equally as well in vitro. The best example where selectivity has been demonstrated is human 14-3-3o. 14-3-3o Preferential homodimerizes with itself and crystallization revealed a structural basis for this isoform s dimerization properties as well as for its specific selectivity for target binding proteins. Here partner specificity is the result of amino acid differences outside of the phosphopeptide-binding cleft. 

This key enzyme of the dissimilatory sulfate reduction was isolated from all Desulfovibrio strains studied until now 135), and from some sulfur oxidizing bacteria and thermophilic Archaea 136, 137). The enzymes isolated from sulfate-reducing bacteria contain two [4Fe-4S] clusters and a flavin group (FAD) as demonstrated by visible, EPR, and Mossbauer spectroscopies. With a total molecular mass ranging from 150 to 220 kDa, APS reductases have a subunit composition of the type 012)32 or 02)3. The subunit molecular mass is approximately 70 and 20 kDa for the a and )3 subunits, respectively. Amino-acid sequence data suggest that both iron-sulfur clusters are located in the (3 subunit... 

Sulfite reductase catalyzes the six-electron reduction of sulfite to sulfide, m essential enzymatic reaction in the dissimilatory sulfate reduction process. Several different types of dissimilatory sulfite reductases were already isolated from sulfate reducers, namely desul-foviridin (148-150), desulforubidin (151, 152), P-582 (153, 154), and desulfofuscidin (155). In addition to these four enzymes, an assimila-tory-type sulfite reductase was also isolated from D. vulgaris. Although all these enzymes have significantly different subunit composition and amino acid sequences, it is interesting to note that, as will be discussed later, all of them share a unique type of cofactor. 

D. desulfuricans is able to grow on nitrate, inducing two enzymes that responsible for the steps of conversion of nitrate to nitrite (nitrate reductase-NAP), which is an iron-sulfur Mo-containing enzyme, and that for conversion of nitrite to ammonia (nitrite reduc-tase-NIR), which is a heme-containing enzyme. Nitrate reductase from D. desulfuricans is the only characterized enzyme isolated from a sulfate reducer that has this function. The enzyme is a monomer of 74 kDa and contains two MGD bound to a molybdenum and one [4Fe-4S] center (228, 229) in a single polypeptide chain of 753 amino acids. FXAFS data on the native nitrate reductase show that besides the two pterins coordinated to the molybdenum, there is a cysteine and a nonsulfur ligand, probably a Mo-OH (G. N. George, personal communication). 

Although the second Cys of the three groups of cysteine residues expected to coordinate the [4Fe-4S] centers of E. coli nitrate reductase were systematically mutated to induce their conversion into [3Fe-4S] centers, this conversion was achieved only in the case of the C247D mutation affecting the lowest potential [4Fe-4S] center (E. Valay, B. Guigliarelli, M. Asso, P. Bertrand, and F. Blasco, unpublished results, 1998). This result, which confirms the proposed coordination scheme (174), shows once again that the coordination capacity of the various Cys motifs of a protein can be differently affected by amino acid substitutions. 

Vitamin K is the cofactor for the carboxylation of glutamate residues in the post-synthetic modification of proteins to form the unusual amino acid y-carboxygluta-mate (Gla), which chelates the calcium ion. Initially, vitamin K hydroquinone is oxidized to the epoxide (Figure 45-8), which activates a glutamate residue in the protein substrate to a carbanion, that reacts non-enzymically with carbon dioxide to form y-carboxyglut-amate. Vitamin K epoxide is reduced to the quinone by a warfarin-sensitive reductase, and the quinone is reduced to the active hydroquinone by either the same warfarin-sensitive reductase or a warfarin-insensitive... 

---