Biochemical and molecular characterization of the extracellular esterase from Streptomyces diastatochromogenes.
An esterase of Streptomyces diastatochromogenes was purified to homogeneity from culture filtrate. The purified enzyme had a molecular mass of 30,862 +/- 5.8 Da, as determined by electrospray mass spectrometry. The esterase-encoding gene was cloned on a 5.1-kb MboI fragment from S. diastatochromogenes genomic DNA into Streptomyces lividans TK23 by using plasmid vector pIJ702. Nucleotide sequence analysis predicted a 978-bp open reading frame, estA, encoding a protein of 326 amino acids, a potential ribosome binding site, and a putative 35- or 36-residue signal peptide for secretion in S. lividans or S. diastatochromogenes, respectively. The transcriptional initiation site was mapped 29 nucleotides upstream from the predicted translational start codon of estA in S. diastatochromogenes. The protein sequence deduced from the estA gene was similar to that of the esterase from the plant pathogen Streptomyces scabies. Both enzymes lacked the conserved motif GXSXG carrying the active-site serine of hydrolytic enzymes. A serine modified by [1,3-3H]diisopropyl fluorophosphate was located at position 11 of the mature enzyme in the sequence GDSYT. This finding and results obtained by site-directed mutagenesis studies indicate that serine 11 may be the active-site nucleophile.
Tesch
C
C
Mikrobielle Genetik, Universität Tübingen, Germany.
Nikoleit
K
K
Gnau
V
V
Götz
F
F
Bormann
C
C
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010944:Plants / Q000382:microbiology; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012694:Serine; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; C098953:EstA protein, bacteria