Gulosibacter molinativorax ON4T molinate hydrolase, a novel cobalt-dependent amidohydrolase

Abstract

A new pathway of molinate mineralization has recently been described. Among the five members of the mixed culture able to promote such a process, Gulosibacter molinativorax ON4(T) has been observed to promote the initial breakdown of the herbicide into ethanethiol and azepane-1-carboxylate. In the current study, the gene encoding the enzyme responsible for molinate hydrolysis was identified and heterologously expressed, and the resultant active protein was purified and characterized. Nucleotide sequence analysis revealed that the gene encodes a 465-amino-acid protein of the metal-dependent hydrolase A subfamily of the amidohydrolase superfamily with a predicted molecular mass of 50.9 kDa. Molinate hydrolase shares the highest amino acid sequence identity (48 to 50%) with phenylurea hydrolases of Arthrobacter globiformis and Mycobacterium brisbanense. However, in contrast to previously described members of the metal-dependent hydrolase A subfamily, molinate hydrolase contains cobalt as the only active-site metal.

Fig. 1.

Transformation of molinate by molinate hydrolase in G. molinativorax ON4^T.

Fig. 2.

Electron microscopic view of purified molinate hydrolase negatively stained with uranyl acetate. (a) An overview, which shows triangular protein masses (circled) to be the dominant molecules. Morphologies indicated by arrows represent biparticulate protein masses of the triangular molecules seen as edge-on views. (b) Detailed view of individual triangular protein masses either focused close to the Gaussian focus (upper row) or at 2.0 μm underfocused (lower row) for maximum contrast and shape discrimination.

Fig. 3.

Evolutionary relationships of members of the metal-dependent hydrolase A subfamily (cd1292) of the amidohydrolase superfamily. The evolutionary histories were inferred using the neighbor-joining method and the p-distance model. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons. Phylogenetic analyses were conducted in MEGA4 (17). Proteins are indicated by their GenBank or PDB accession number and the host strain. The bar represents 0.05 amino acid difference per site. Proteins of validated function are indicated by filled circles, proteins not sharing the His-His-Lys-His-His-Asp metal-binding motif are indicated by filled diamonds, and the protein of unknown function where a crystal structure is available is indicated by an open diamond.

Fig. 4.

Multiple-sequence alignment of protein regions of molinate hydrolase and selected members of the metal-dependent hydrolase A subfamily (cd1292) of the amidohydrolase superfamily. Putative metal-binding residues, typically of a His-His-Lys-His-His-Asp motif, are boxed. C., Caulobacter; M., Microbacterium; So., Sorangium; Sh., Shewanella.