Sridhar Prasad G, Kresge N, Muhlberg A B, Shaw A, Jung Y S, Burgess B K, Stout C D
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037-1093, USA.
Protein Sci. 1998 Dec;7(12):2541-9. doi: 10.1002/pro.5560071207.
ferredoxin reductase (AvFPR) is involved in the response to oxidative stress in Azotobacter vinelandii. The crystal structure of AvFPR has been determined at 2.0 A resolution. The polypeptide fold is homologous with six other oxidoreductases whose structures have been solved including Escherichia coli flavodoxin reductase (EcFldR) and spinach, and Anabaena ferredoxin:NADP+ reductases (FNR). AvFPR is overall most homologous to EcFldR. The structure is comprised of a N-terminal six-stranded antiparallel beta-barrel domain, which binds FAD, and a C-terminal five-stranded parallel beta-sheet domain, which binds NADPH/NADP+ and has a classical nucleotide binding fold. The two domains associate to form a deep cleft where the NADPH and FAD binding sites are juxtaposed. The structure displays sequence conserved motifs in the region surrounding the two dinucleotide binding sites, which are characteristic of the homologous enzymes. The folded over conformation of FAD in AvFPR is similar to that in EcFldR due to stacking of Phe255 on the adenine ring of FAD, but it differs from that in the FNR enzymes, which lack a homologous aromatic residue. The structure of AvFPR displays three unique features in the environment of the bound FAD. Two features may affect the rate of reduction of FAD: the absence of an aromatic residue stacked on the isoalloxazine ring in the NADPH binding site; and the interaction of a carbonyl group with N10 of the flavin. Both of these features are due to the substitution of a conserved C-terminal tyrosine residue with alanine (Ala254) in AvFPR. An additional unique feature may affect the interaction of AvFPR with its redox partner ferredoxin I (FdI). This is the extension of the C-terminus by three residues relative to EcFldR and by four residues relative to FNR. The C-terminal residue, Lys258, interacts with the AMP phosphate of FAD. Consequently, both phosphate groups are paired with a basic group due to the simultaneous interaction of the FMN phosphate with Arg51 in a conserved FAD binding motif. The fourth feature, common to homologous oxidoreductases, is a concentration of 10 basic residues on the face of the protein surrounding the active site, in addition to Arg51 and Lys258.
铁氧化还原蛋白还原酶（AvFPR）参与了棕色固氮菌对氧化应激的反应。已确定AvFPR的晶体结构分辨率为2.0埃。该多肽折叠与其他六种已解析结构的氧化还原酶同源，包括大肠杆菌黄素氧化还原蛋白还原酶（EcFldR）、菠菜和鱼腥藻铁氧化还原蛋白：NADP +还原酶（FNR）。AvFPR总体上与EcFldR同源性最高。该结构由一个结合FAD的N端六链反平行β桶结构域和一个结合NADPH / NADP +且具有经典核苷酸结合折叠的C端五链平行β片层结构域组成。这两个结构域结合形成一个深裂隙，NADPH和FAD结合位点并列于此。该结构在两个二核苷酸结合位点周围的区域显示出序列保守基序，这是同源酶的特征。由于Phe255堆积在FAD的腺嘌呤环上，AvFPR中FAD的折叠构象与EcFldR中的相似，但与FNR酶不同，后者缺乏同源芳香族残基。AvFPR的结构在结合FAD的环境中显示出三个独特特征。两个特征可能影响FAD的还原速率：NADPH结合位点中缺乏堆积在异咯嗪环上的芳香族残基；以及一个羰基与黄素的N10相互作用。这两个特征都是由于AvFPR中保守的C端酪氨酸残基被丙氨酸（Ala254）取代所致。另一个独特特征可能影响AvFPR与其氧化还原伙伴铁氧化还原蛋白I（FdI）的相互作用。这是C端相对于EcFldR延伸了三个残基，相对于FNR延伸了四个残基。C端残基Lys258与FAD的AMP磷酸基团相互作用。因此，由于FMN磷酸基团与保守的FAD结合基序中的Arg51同时相互作用，两个磷酸基团都与一个碱性基团配对。第四个特征是同源氧化还原酶共有的，除了Arg51和Lys258外，在围绕活性位点的蛋白质表面还有10个碱性残基集中分布。
