Summary Histidine phosphorylation (pHis) is well studied in bacteria; however, its role in mammalian signaling remains largely unexplored due to the lack of pHis-specific antibodies and the lability of the phosphoramidate (P-N) bond. Both imidazole nitrogens can be phosphorylated, forming 1-phosphohistidine (1-pHis) or 3-phosphohistidine (3-pHis). We have developed monoclonal antibodies (mAbs) that specifically recognize 1-pHis or 3-pHis; they do not cross-react with phosphotyrosine or the other pHis isomer. Assays based on the isomer-specific auto-phosphorylation of NME1 and phosphoglycerate mutase were used with immunoblotting and sequencing IgG variable domains to screen, select and characterize anti-1-pHis and anti-3-pHis mAbs. Their sequence independence was determined by blotting synthetic peptide arrays, and they have been tested for immunofluorescence staining and immunoaffinity purification, leading to putative identification of pHis-containing proteins. These reagents should be broadly useful for identification of pHis substrates and functional study of pHis using a variety of immunological, proteomic and biological assays.

A new type of nitrilase, arylacetonitrilase, has been purified from isovaleronitrile-induced cells of Alcaligenes faecalis JM3 in four steps. The purity of the enzyme was confirmed by SDS/polyacrylamide gel electrophoresis, ampholyte electrofocusing and double immunodiffusion in agarose. The enzyme has a molecular mass of about 275 kDa and consists of six subunits of identical molecular mass. The purified enzyme exhibits a pH optimum of 7.5 and a temperature optimum of 45 degrees C. The enzyme is specific for arylacetonitriles such as 2-thiophenacetonitrile, p-tolyacetonitrile, p-chlorobenzylcyanide, p-fluorobenzylcyanide and 3-pyridylacetonitrile. The enzyme stoichiometrically catalyzes the hydrolysis of arylacetonitrile to arylacetic acid and ammonia, no formation of amide occurring. However, the enzyme does not attack nitrile groups attached to aromatic and heteroaromatic rings, which are hydrolyzed preferably by the nitrilases known previously. The enzyme requires thiol compounds such as dithiothreitol and 2-mercaptoethanol to exhibit its maximum activity.

Nitrile hydratase, which occurs abundantly in cells of Rhodococcus rhodochrous Jl isolated from soil samples, catalyzes the hydration of 3-cyanopyridine to nicotinamide. By using resting cells, the reaction conditions for nicotinamide production were optimized. Under the optimum conditions, 100% of the added 12 M 3-cyanopyridine was converted to nicotinamide without the formation of nicotinic acid, and the highest yield achieved was 1,465 g of nicotinamide per liter of reaction mixture containing resting cells (1.48 g as dry cell weight) in 9 h. The nicotinamide produced was crystallized and then identified physicochemically. The further conversion of the nicotinamide to nicotinic acid was due to the low activity of nicotinamide as a substrate for the amidase(s) present in this organism. Nitrile hydratase catalyzes the hydration of nitriles to amides, whereas nitrilase catalyzes the direct cleavage of nitriles to the corresponding acids and ammonia. Thus, nitrile hydratase can be clearly distinguished from nitrilase by the mode of nitrile degradation. We previously proposed a new enzymatic production process for acrylamide, applicable on an industrial scale, involving nitrile hydratase as a catalyst (3). Pseudomonas chlororaphis B23 and Brevibacterium sp. strain R312 were selected as favorable strains (3, 6). Indeed, P. chlororaphis B23 produces more than 400 g of acrylamide per liter of reaction mixture from acrylonitrile under suitable conditions. Thus, very recently, the use of the P. chlororaphis B23 nitrile hydratase as a catalyst for the industrial production of acrylamide, an important chemical commodity, was started. Recently, we found that Rhodococcus rhodochrous J1 produces nitrile hydratase or nitrilase selectively, depending on the culture conditions (T. Nagasawa, M. Kobayashi, and H. Yamada, Arch. Microbiol., in press). Although the nitrile

The Aurora family of serine/threonine kinases is essential for mitosis. Their crucial role in cell cycle regulation and aberrant expression in a broad range of malignancies have been demonstrated and have prompted intensive search for small molecule Aurora inhibitors. Indeed, over 10 of them have reached the clinic as potential anticancer therapies. We report herein the discovery and optimization of a novel series of tricyclic molecules that has led to SAR156497, an exquisitely selective Aurora A, B, and C inhibitor with in vitro and in vivo efficacy. We also provide insights into its mode of binding to its target proteins, which could explain its selectivity.

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