The primary aim of this study was to decipher the catalytic functions of the NHase with wide substrate spectra from Rhodococcus ruber CGMCC3090 by computer modeling and substrate docking. 3D structure model of the enzyme was built by computer modeling to obtain the optimal structure. The larger binding site cavity (559 angstrom(3)) indicated that this NHase may catalyze a large variety of substrates of nitrites. Some key residues such as alpha Glu82, alpha Gln83, beta Tyr71, beta Tyr72, beta Arg52 and beta Arg55 surrounding the binding site were unique compared with those of 3QXE as a template, indicating that the enzyme may have unusual substrate specificity. The docking and the biotransformation experiments demonstrated that the special docking pose and shorter distance proved to be more effective for the enzyme to improve function. (c) 2012 Elsevier Inc. All rights reserved.