Discovery of a Novel Enzyme, Isonitrile Hydratase, Involved in Nitrogen-Carbon Triple Bond Cleavage

Abstract

Isonitrile containing an NϵC triple bond was degraded by microorganism sp. N19-2, which was isolated from soil through a 2-month acclimatization culture in the presence of this compound. The isonitrile-degrading microorganism was identified as Pseudomonas putida. The microbial degradation was found to proceed through an enzymatic reaction, the isonitrile being hydrated to the corresponding N-substituted formamide. The enzyme, named isonitrile hydratase, was purified and characterized. The native enzyme had a molecular mass of about 59 kDa and consisted of two identical subunits. The enzyme stoichiometrically catalyzed the hydration of cyclohexyl isocyanide (an isonitrile) to N-cyclohexylformamide, but no formation of other compounds was detected. The apparent K m value for cyclohexyl isocyanide was 16.2 mM. Although the enzyme acted on various isonitriles, no nitriles or amides were accepted as substrates.

Isonitrile Hydratase from Pseudomonas putidaN19–2

“…Strains, Plasmids, and Media-P. putida N19 -2 was isolated previously from soil (20). E. coli DH10B (Invitrogen) was used as the host for pUC plasmids (23).…”

“…N19 -2, from soil and identified it as Pseudomonas putida. In this strain, we discovered a novel enzyme that catalyzes the hydration of an isonitrile to the corresponding N-substituted formamide and named it isonitrile hydratase (20). It has been approved as a new enzyme by NC-IUBMB; EC 4.2.1.103.…”

Hyper-inducible expression system for streptomycetes

“…The amplified fragment was inserted into HindIII-PstI-digested pSH19 to generate nitA::pSH19. In like manner, inhA (22,23) and xylE (24,25) were also amplified by PCR using in each case a primer pair which contained a flanking HindIII site within the forward primer (SDinhA-FR or SDxylE-FR) and a BamHI site within the reverse primer (inhA-RV or xylE-RV), respectively. Each amplified fragment was inserted into HindIII-BamHI-digested pSH19 to generate inhA::pSH19 and xylE::pSH19, respectively.…”

“…Nitrilase (15,17), isonitrile hydratase (22,23), and catechol 2,3-dioxygenase (24, 25) activities were assayed by described methods. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the formation of 1 mol of product per min from the substrate under the standard experimental conditions.…”

“…To evaluate the P nitA -NitR system as an inducible expression system, three genes [i.e., nitA (15,17), inhA (22,23), and xylE (24,25)] were used for heterologous protein expression. inhA and xylE are derived from Pseudomonas sp.…”