Isonitrile Hydratase from Pseudomonas putidaN19–2

Abstract

Isonitrile hydratase is a novel enzyme in Pseudomonas putida N19 -2 that catalyzes the conversion of isonitriles to N-substituted formamides. Based on N-terminal and internal amino acid sequences, a 535-bp DNA fragment corresponding to a portion of the isonitrile hydratase gene was amplified, which was used as a probe to clone a 6.4-kb DNA fragment containing the whole gene. Sequence analysis of the 6.4-kb fragment revealed that the isonitrile hydratase gene (inhA) was 684 nucleotides long and encoded a protein with a molecular mass of 24,211 Da. Overexpression of inhA in Escherichia coli gave a large amount of soluble isonitrile hydratase exhibiting the same molecular and catalytic properties as the native enzyme from the Pseudomonas strain. The predicted amino acid sequence of inhA showed low similarity to that of an intracellular protease in Pyrococcus horikoshii (PH1704), and an active cysteine residue in the protease was conserved in the isonitrile hydratase at the corresponding position (Cys-101). A mutant enzyme containing Ala instead of Cys-101 did not exhibit isonitrile hydratase activity at all, demonstrating the essential role of this residue in the catalytic function.

Evolution of New Enzymatic Function by Structural Modulation of Cysteine Reactivity in Pseudomonas fluorescens Isocyanide Hydratase

“…Similar to other DJ-1 superfamily proteins, ICH requires Cys 101 for enzymatic activity (9). The active site environment of Cys 101 is dominated by hydrogen bonding to residues Asp 17 and Thr 102 (Fig.…”

“…1B). Previous enzymological studies of ICH have shown that catalysis requires a conserved cysteine residue (Cys 101 ) in the active site, the enzyme is highly selective for isocyanides and will not catalyze the hydrolysis of the corresponding nitrile compounds, and the enzyme has a broad substrate tolerance for organic isocyanides (8,9). ICH is interesting because, in addition to catalyzing the hydration of an unusual class of compounds, it is also a new and poorly characterized member of the large DJ-1 superfamily.…”

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.…”