Nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a novel enzyme that possesses a non-heme iron(III) center binding endogenous nitric oxide (NO). It is inactivated by aerobic incubation of cells in the dark, whereas the inactive form is converted to the active one by light irradiation. To clarify the mechanism of activity regulation in the NHase, we investigated the role of NO. When the inactive NHase was irradiated in the presence of Fe(II) ions and a spin trap, N-methyl-D-glucamine dithiocarbamate (MGD), three hyperfine lines from the nitrogen atom of the [(MGD) 2 -Fe II -NO] complex were resolved, indicating that NO was released from the enzyme upon photoactivation. The amount of NO release was obtained as 0.99 ( 0.12 per enzyme (n ) 4) by using an NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide. The active NHase was completely inactivated by exogenous NO, and upon photoirradiation 86% of the original activity was restored. FTIR, ESR, and UV-VIS absorption measurements confirmed the view that association of NO restores the original inactive form of the enzyme. The rate constant for nitrosylation of the enzyme activated by a laser pulse asymptotically increased with an increase in NO concentration. A kinetic analysis of the NHase nitrosylation demonstrated that inactivation of the enzyme by NO binding proceeded via an intermediate. The rate constant for inactivation and the quantum yield of photoactivation were determined as 14 s -1 and 0.48, respectively. It is thus concluded that the activity of the NHase is regulated by nitrosylation and photoinduced denitrosylation of the non-heme iron center. This finding provides a new aspect regarding biological function of NO, i.e., regulation of the enzymatic activity with the aid of light.

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We performed random sequencing of cDNAs from nine biologically or industrially important cultures of the industrially valuable fungus Aspergillus oryzae to obtain expressed sequence tags (ESTs). Consequently, 21 446 raw ESTs were accumulated and subsequently assembled to 7589 non-redundant consensus sequences (contigs). Among all contigs, 5491 (72.4%) were derived from only a particular culture. These included 4735 (62.4%) singletons, i.e. lone ESTs overlapping with no others. These data showed that consideration of culture grown under various conditions as cDNA sources enabled efficient collection of ESTs. BLAST searches against the public databases showed that 2953 (38.9%) of the EST contigs showed significant similarities to deposited sequences with known functions, 793 (10.5%) were similar to hypothetical proteins, and the remaining 3843 (50.6%) showed no significant similarity to sequences in the databases. Culture-specific contigs were extracted on the basis of the EST frequency normalized by the total number for each culture condition. In addition, contig sequences were compared with sequence sets in eukaryotic orthologous groups (KOGs), and classified into the KOG functional categories.