Purification and characterization of Pseudomonas putida histidine ammonia-lyase expressed in Escherichia coli.
Histidine ammonia-lyase (HAL) from Pseudomonas putida PRS1 contains a catalytically important electrophilic center reported to be dehydroalanine. Little is known about the origin of this group or its linkage to the protein. To initiate structural studies on this enzyme, P. putida HAL was purified from an Escherichia coli high-expression clone in which the HAL gene (hutH) was under the control of the lambda PL promoter on a plasmid vector. In this clone from 6 to 10% of the soluble cell protein after heat induction was HAL and approximately 200 mg of 95% pure HAL could be obtained from 120 g wet weight of cells in a 40 to 60% yield. The overexpressed protein was identical to P. putida HAL in native molecular weight (220 kDa), subunit composition (four identical subunits of 53 kDa each), affinity for substrate (L-histidine Km of 5.3 mM at pH 9.0), and its sensitivity to inactivation by cyanide and bisulfite. The N-terminal amino acid sequence was in agreement with the DNA-predicted sequence, indicating proper translational initiation. These features make this enzyme an appropriate candidate for protein structure investigations regarding the nature of the electrophilic center and its association with the protein.
Hernandez
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Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Phillips
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002384:Catalysis; D002412:Cations / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli; D015870:Gene Expression; D006638:Histidine Ammonia-Lyase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D015202:Protein Engineering; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D001426:Bacterial Proteins; D002412:Cations; D011993:Recombinant Fusion Proteins; D006638:Histidine Ammonia-Lyase