Characterization and Regulation of the gbuA Gene, Encoding Guanidinobutyrase in the Arginine Dehydrogenase Pathway of Pseudomonas aeruginosa PAO1

Abstract

ABSTRACT The arginine dehydrogenase (or oxidase) pathway catabolically converts arginine to succinate via 2-ketoglutarate and 4-guanidinobutyrate (4-GB) with the concomitant formation of CO 2 and urea. Guanidinobutyrase (GBase; EC 3.5.3.7) catalyzes the conversion of 4-guanidinobutyrate to 4-aminobutyrate and urea in this pathway. We investigated the structure and regulation of the gene for GBase (designated gbuA ) of Pseudomonas aeruginosa PAO1 and characterized the gbuA product. The gbuA and the adjacent gbuR genes were cloned by functional complementation of a gbuA9005 mutant of strain PAO1 defective in 4-GB utilization. The deduced amino acid sequence of GbuA (319 amino acids; M r 34,695) assigned GBase to the arginase/agmatinase family of C-N hydrolases. Purified GbuA was a homotetramer of 140 kDa that catalyzed the specific hydrolysis of 4-GB with K m and K cat values of 49 mM and 1,012 s −1, respectively. The divergent gbuR gene, which shared the intergenic promoter region of 206 bp with gbuA , encoded a putative regulatory protein (297 amino acids; M r 33,385) homologous to the LysR family of proteins. Insertional inactivation of gbuR by a gentamicin resistance cassette caused a defect in 4-GB utilization. GBase and gbuA ′::′ lacZ fusion assays demonstrated that this gbuR mutation abolishes the inducible expression of gbuA by exogenous 4-GB, indicating that GbuR participates in the regulation of this gene. Northern blotting located an inducible promoter for gbuA in the intergenic region, and primer extension localized the transcription start site of this promoter at 40 bp upstream from the initiation codon of gbuA . The gbuRA genes at the genomic map position of 1547000 are unlinked to the 2-ketoarginine utilization gene kauB at 5983000, indicative of at least two separate genetic units involved in the arginine dehydrogenase pathway.