Nitrate reductase from the magnetotactic bacterium Magnetospirillum magnetotacticum MS-1: purification and sequence analyses

金沢大学理学部金沢大学大学院自然科学研究科We purified the nitrate reductase from the soluble fraction of Magnetospirillum magnetotacticum MS-1. The enzyme was composed of 86- and 17-kDa subunits and contained molybdenum, non-heme iron, and heme c. These properties are very similar to those of the periplasmic nitrate reductase found in Paracoccus pantotrophus. The M. magnetotacticum nap locus was clustered in seven open reading frames, napFDAGHBC. The phylogenetic analyses of NapA, NapB, and NapC suggested a close relationship between M. magnetotacticum nap genes and Escherichia coli nap genes, which is not consistent with the 16S rDNA data. This is the first finding that the α subclass of Proteobacteria possesses a napFDAGHBC-type nap gene cluster. The nap gene cluster had putative fumarate and nitrate reduction regulatory protein (Fnr) and NarL protein binding sites. Furthermore, we investigated the effect of molybdate deficiency in medium on the total iron content of the magnetosome fraction and discussed the physiological function of nitrate reductase in relation to the magnetite synthesis in M. magnetotacticum

Purification and characterization of dissimilatory nitrate reductase from a denitrifying halophilic archaeon, Haloarcula marismortui

AbstractDissimilatory nitrate reductase was purified from a denitrifying halophilic archaeon, Haloarcula marismortui, to an electrophoretically homogeneous state. The purified enzyme was inferred to be a homotetramer composed of a 63 kDa polypeptide. The electron paramagnetic resonance spectrum of the purified enzyme revealed typical rhombic signals which were ascribed to Mo(V) in the Mo–molybdopterin complex. Like the bacterial membrane-bound (Nar-) enzyme, the purified enzyme supported the catalysis of chlorate. The enzyme was activated in extreme saline conditions and the values of kcat and Km toward nitrate were 145 s−1 and 79 μM, respectively, in the presence of 2.0 M NaCl

Expression and purification of cyto-insectotoxin (Cit1a) using silkworm larvae targeting for an antimicrobial therapeutic agent

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Cdc37 Interacts with the Glycine-Rich Loop of Hsp90 Client Kinases

Recently, we identified a client-binding site of Cdc37 that is required for its association with protein kinases. Phage display technology and liquid chromatography-tandem mass spectrometry (which identifies a total of 33 proteins) consistently identify a unique sequence, GXFG, as a Cdc37-interacting motif that occurs in the canonical glycine-rich loop (GXGXXG) of protein kinases, regardless of their dependence on Hsp90 or Cdc37. The glycine-rich motif of Raf-1 (GSGSFG) is necessary for its association with Cdc37; nevertheless, the N lobe of Raf-1 (which includes the GSGSFG motif) on its own cannot interact with Cdc37. Chimeric mutants of Cdk2 and Cdk4, which differ sharply in their affinities toward Cdc37, show that their C-terminal portions may determine this difference. In addition, a nonclient kinase, the catalytic subunit of cyclic AMP-dependent protein kinase, interacts with Cdc37 but only when a threonine residue in the activation segment of its C lobe is unphosphorylated. Thus, although a region in the C termini of protein kinases may be crucial for accomplishing and maintaining their interaction with Cdc37, we conclude that the N-terminal glycine-rich loop of protein kinases is essential for physically associating with Cdc37