The crystal structure of a major oxygen-insensitive nitroreductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-Å resolution. This enzyme is a homodimeric flavoprotein with one FMN cofactor per monomer and catalyzes reduction of nitrocompounds using NADPH. The structure exhibits an ␣ ؉ ␤-fold, and is comprised of a central domain and an excursion domain. The overall structure of NfsA is similar to the NADPH-dependent flavin reductase of Vibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site. Nitroaromatic compounds including nitrofurans, nitropyrenes, and nitrobenzenes have been used as antimicrobial agents, food additives and raw materials in several industrial processes (1-5), and as a result are distributed widely around the environment. Many of these compounds are toxic, mutagenic, or carcinogenic (6 -8). It is believed that enzymatic transformation is needed for nitroaromatic compounds to show these serious effects (9, 10). The reduction of a nitro group of a parent nitrocompound is a key step of this process (11,12). Enzymes, which catalyze the reduction of nitrocompounds using a reduced pyridine nucleotide, are termed nitroreductases and are distinguished by their sensitivity of activity to oxygen (9, 10).The oxygen-sensitive enzymes can catalyze nitroreduction only under anaerobic conditions. A nitro-anion radical formed by a one-electron transfer is immediately reoxidized in the presence of oxygen to a parent nitrocompound and superoxide (13,14). In this futile cycle, reducing equivalents are consumed without the progress of nitroreduction and nitrocompounds perform as a catalyst to reduce oxygen. On the other hand, the oxygen-insensitive enzymes catalyze an obligatory two-electron reduction. A nitro group of a parent nitrocompound is reduced by a series of two-electron transfers, through nitroso and hydroxylamine intermediates, and finally to an amino group (13). The hydroxylamine intermediate arising from the four-electron transfer in total is found to be toxic, carcinogenic, or mutagenic.Three proteins with oxygen-insensitive nitroreductase activity in Escherichia coli have been identified (15). NfsA 1 is the major component, while NfsB and NfsC are minor components. NfsA and NfsB have been well studied relative to NfsC. NfsA and NfsB have similar enzymatic property, although NfsA has only 7% identity with NfsB on the amino acid sequence alignment. Both NfsA and NfsB are flavoenzymes with FMN as the prosthetic group and catalyze the reduction of nitrocompounds by Ping Pong Bi Bi kinetics (16,17). Counterparts of NfsA and NfsB, found in luminescent bacteria (16,17), are flavin reductase (FRP) of Vibrio harveyi (18) and flavin reductase (FRase I) of Vibrio fischeri (19), respectively. Enzyme that resembles FRP in the amino acid sequence alignment is also found in Bacillus subtilus and is called NfrA1 (20). A comparison of * This work was supported in part by grants-in-aid for Scientific Res...

No abstract

There is functional evidence that polycystin-2 (TRPP2) interacts with other members of the transient receptor potential family, including TRPC1 and TRPV4. Here we have used atomic force microscopy to study the structure of the TRPP2 homomer and the interaction between TRPP2 and TRPC1. The molecular volumes of both Myc-tagged TRPP2 and V5-tagged TRPC1 isolated from singly transfected tsA 201 cells indicated that they assembled as homotetramers. The molecular volume of the protein isolated from cells expressing both TRPP2 and TRPC1 was intermediate between the volumes of the two homomers, suggesting that a heteromer was being formed. The distribution of angles between pairs of anti-Myc antibodies bound to TRPP2 particles had a large peak close to 90°and a smaller peak close to 180°, consistent with the assembly of TRPP2 as a homotetramer. In contrast, the corresponding angle distributions for decoration of the TRPP2-TRPC1 heteromer by either anti-Myc or anti-V5 antibodies had predominant peaks close to 180°. This decoration pattern indicates a TRPP2:TRPC1 subunit stoichiometry of 2:2 and an alternating subunit arrangement.Autosomal dominant polycystic kidney disease is one of the commonest inherited human disorders (reviewed in Ref. 1). It has a population prevalence of over 1:1,000 in all ethnic groups and is a leading cause of end stage renal failure. Autosomal dominant polycystic kidney disease is characterized by the progressive loss of normal renal parenchyma secondary to the development of multiple fluid-filled cysts derived from renal tubular epithelial cells. It is caused by mutations in two genes, PKD1 and PKD2, whose protein products, polycystin-1 (2, 3) and polycystin-2 (or TRPP2) (4) form a Ca 2ϩ -permeable ion channel complex (5). This complex transduces extracellular mechanical stimuli via the renal primary cilium (6) and regulates multiple intracellular Ca 2ϩ -sensitive signaling pathways (5, 7). TRPP2 also appears to have a role, independent of polycystin-1, in regulating Ca 2ϩ efflux from the endoplasmic reticulum (8, 9). In addition to its interaction with polycystin-1, TRPP2 is known to interact with other members of the TRP 4 superfamily, such as TRPC1 (10) and TRPV4 (11), raising the question of the architecture of these heteromeric complexes.TRP channel complexes have been assumed to be tetramers, initially on the basis of the resemblance of the primary structure of the TRP channel subunits to that of the Shaker K ϩ channel, which is known to be tetrameric (12). Moreover, a variety of structural and functional techniques have been used to demonstrate a tetrameric structure for a number of TRP channel family members, including TRPC1 (13), TRPC3 (14), TRPV1 (15, 16), TRPV5 and TRPV6 (17), and TRPM2 (18). Intriguingly, data have recently been presented indicating that TRPP2 exists in the plasma membrane as a trimer, which is then able to interact with polycystin-1 to form a heteromer with a 3:1 stoichiometry (19). It is puzzling that TRPP2 appears to behave differently from all other TRP channel...

ipa-43d is a hypothetical gene identified by the Bacillus subtilis genome project (Mol. Microbiol. 10, 371-384 1993; Nature 390, 249-256 1997). The ipa-43d protein overexpressed in E. coli was purified to homogeneity and its properties were analyzed biochemically. The ipa-43d protein was found to be tightly associated with FMN and to be capable of reducing both nitrofurazone and FMN effectively. Although the ipa-43d protein catalysis obeys the ping-pong Bi-Bi mechanism, catalysis mode was changed to the sequential mechanism upon coupling with the bioluminescent reaction. Database search showed that B. subtilis possessed four genes (ipa-44d, ytmO, yddN, and yvbT), encoding proteins similar in amino acid sequence to LuxA and LuxB of Photobacterium fischeri, and, in particular, ipa-44d is immediately adjacent to the ipa-43d gene on the chromosome.