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P E Stevis

Publications and source records attributed to P E Stevis.

6 recordsLinked to original sources
PubMed1992

Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase.

Site-directed mutagenesis was used to examine the catalytic importance of 2 histidine and 4 arginine residues in Escherichia coli periplasmic acid phosphatase (EcAP). The residues that were selected as targets for mutagenesis were those that were also conserved in a number of high molecular weight acid phosphatases from eukaryotic organisms, including human prostatic and lysosomal acid phosphatases. Both wild type EcAP and mutant proteins were overproduced in E. coli using an expression system based on the T7 RNA polymerase promoter, and the proteins were purified to homogeneity. Examination of the purified mutant proteins by circular dichroism and proton NMR spectroscopy revealed no significant conformational changes. The replacement of Arg16 and His17 residues that were localized in a conserved N-terminal RHGXRXP motif resulted in the complete elimination of EcAP enzymatic activity. Critical roles for Arg20, Arg92, and His303 were also established because the corresponding mutant proteins exhibited residual activities that were not higher than 0.4% of that of wild type enzyme. In contrast, the replacement of Arg63 did not cause a significant alteration of the kinetic parameters. The results are in agreement with a previously postulated distant relationship between acid phosphatases, phosphoglycerate mutases, and fructose-2,6-bisphosphatase. These and earlier results are also consistent with the conclusion that 2 histidine residues participate in the catalytic mechanism of acid phosphatases, with His17 playing the role of a nucleophilic acceptor of the phospho group, whereas His303 may act as a proton donor to the alcohol or phenol.

Acid Phosphatase↗
PubMed1991

Covalent structure, disulfide bonding, and identification of reactive surface and active site residues of human prostatic acid phosphatase.

The pairing of the half-cysteine residues of human prostatic acid phosphatase was established by proteolytic digestion and analysis of the resulting peptide mixtures by fast atom bombardment mass spectrometry (FAB-MS). An independently derived, full length cDNA clone was used as the basis for the interpretation of the FAB-MS data. The sequence of the native protein is that predicted from the present cDNA sequence, except for the carboxyl-terminal end and some possible post-translational deamidations. Isolated human prostatic acid phosphatase was found to have multiple carboxyl-terminal ends, terminating in Thr, Glu, and Asp, corresponding to residues 349-351 of the 354-residue protein that is predicted from the cDNA sequence after removal of a leader peptide. The protein contains no free sulfhydryl groups. The identical monomer chains of the dimeric native enzyme are found to contain three disulfide bonds, specifically Cys-129 to Cys-340, Cys-183 to Cys-281, and Cys-315 to Cys-319. In view of the conserved positions of cysteines in the homologous human and rat liver lysosomal acid phosphatases, an identical disulfide bonding pattern may be predicted for those proteins. The location of a potential antigenic site was established by selective labeling of proximate tyrosine residues predicted to be on the surface. A conserved RHGXRXP sequence is present in the prostatic, lysosomal, Escherichia coli, and yeast acid phosphatases and is predicted to be of mechanistic significance. In addition, residue Arg-54 is shown to be an active site residue by reaction of the enzyme with phenylglyoxal. Interestingly, this residue is present in a sequence RXRY (R,H) that is also present in lysosomal phosphatase and in recently described protein tyrosine phosphatases.

Acid Phosphatase↗
PubMed1988

A novel xylB-based positive selection vector.

Expression of a plasmid-borne Escherichia coli xylulokinase gene (xylB) under the control of the lac promoter yields constitutively high levels of xylulokinase activity. When a plasmid containing this lac-xylB fusion (pLEK100) is transformed into a xylB- mutant the Xyl+ phenotype is restored on xylose-containing media. When the same transformants are plated on xylitol medium, growth inhibition is observed. Positive selection is achieved by cloning DNA into the unique restriction sites of pLEK100, to disrupt xylB expression, transforming E. coli, and then plating transformants on xylitol medium. With this protocol only transformants with insert containing plasmids will be obtained. This results in a considerable reduction in the time and effort needed to construct genomic libraries or perform routine DNA cloning experiments. Three unique sites are available which are suitable for positive selection of DNA fragments, via the disruption of translation (BglII) or transcription (HindIII, SalI, and BglII) of the xylB gene.

Escherichia coli↗
PubMed1987

Positive selection vectors based on xylose utilization suppression.

High levels of xylose isomerase activity in wild-type Escherichia coli strains results in a Xyl- phenotype. This phenomenon was exploited for the development of a versatile positive selection system. The xylA promoter was deleted with the exonuclease BAL 31 and the resulting structural gene was inserted into the SmaI site of pUC9, yielding the prototype vector, pLX100. In this construct xylA expression is placed under the transcriptional control of the lac promoter. Transformation of any wild-type E. coli strain with pLX100 results in high levels of xylose isomerase and a Xyl- phenotype. Decreasing the activity below a critical level (approx. 100 u) restores the Xyl+ phenotype. pLX100 contains contiguous restriction sites for HindIII, PstI, BamHI and XhoI, suitable for positive selection cloning experiments. E. coli transformants containing pLX100 cannot grow in minimal medium with xylose unless a DNA fragment is inserted into any one of the unique restriction sites. This makes the plasmid an ideal positive-selection cloning vector.

Aldose-Ketose Isomerases↗
PubMed1984

Cloning and characterization of the xyl genes from Escherichia coli.

Specific xylose utilization mutants of Escherichia coli were isolated that had altered xylose isomerase ( xylA ), xylulokinase ( xylB ), and regulatory ( xylR ) or transport ( xylT ) activities. We screened the Clarke and Carbon E. coli gene bank and one clone, pLC10 -15, was found to complement the xyl mutants we had characterized. Subcloning and DNA restriction mapping allowed us to locate the xylA and xylB genes on a 1.6 kbp Bg/II fragment and a 2.6 kbp HindIII-Sa/I fragment, respectively. The identification and mapping of xyl gene promoters suggest that the xylA and xylB genes are organized as an operon having a single xylose inducible promoter preceding the xylA gene.

Aldose-Ketose Isomerases↗