Discovery of a novel (+)-γ-lactamase from Bradyrhizobium japonicum USDA 6 by rational genome mining

Abstract

A novel (+)-γ-lactamase used for the resolution of racemic γ-lactam from Bradyrhizobium japonicum USDA 6 was found as a result of sequence-structure guided genome mining. It consists of 409 amino acids, only 49% of which are identical to the amino acid sequences of the known (+)-γ-lactamase from Sulfolobus solfataricus. This is only the third (+)-γ-lactamase gene to be reported.

Fig 1

Enzymatic preparation of (−)-γ-lactam for the synthesis of abacavir.

Fig 2

Multiple alignment of (+) γ-lactamase sequence and homologous enzymes. Abbreviations: S. solfataricus, amidase from Sulfolobus solfataricus (accession number NP_343511); R.spN771, amidase from Rhodococcus sp. N771 (Protein Data Bank [PDB] 3A1K_A); P. fluorescens, amidase from Pseudomonas fluorescens (accession number ZP_07774754); B. japonicum, amidase from Bradyrhizobium japonicum (accession number YP_005605100). Helices are represented by large dark-gray cylinders, β-strands by arrows, and coils by lines. A dashed line represents gaps in the amino acid sequence alignment.

Fig 3

Coomassie-stained gel after SDS-PAGE analysis of expressed proteins from transformed bacteria using gentle lysis. M, protein marker; lane 1, E. coli BL21(DE3) cells transformed with empty pET-30a vector; lane 2, E. coli BL21(DE3) cells transformed with pET-30a-(+)-γ-lactamase vector.

Fig 4

Chiral HPLC analysis of the resolution.