Abstract/Details

YZGD pyridoxal phosphatase from Paenibacillus thiaminolyticus; subcloning, expression, and purification for x-ray crystallography structure determination


2008 2008

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Abstract (summary)

The HAD superfamily contains enzymes that catalyze carbon or phosphate transfer reactions. One family within the HAD superfamily is the p-nitrophenyl phosphatase (PNPPase) family. Members of this family are all phosphatases that cleave the substrate analog p-nitrophenyl phosphate, in addition to their biologically significant substrate. Interestingly, among the small number of family members that have been characterized, there is a wide variety of biologically relevant activities. YZGD from Paenibacillus thiaminolyticus has pyridoxal phosphatase activity, catalyzed by a HAD superfamily domain, as well as a CDP-alcohol/sugar nucleotide hydrolase activity, catalyzed by a Nudix hydrolase domain. Determining the structure of this enzyme would result in valuable information to contribute to our knowledge and understanding of the HAD superfamily and the Nudix hydrolase superfamily. Sufficient quantities of sufficiently pure YZGD are necessary to perform x-ray crystal structure determination. A histidine tag was added to the enzyme so that it can be purified by nickel affinity chromatography. This was done by cloning the gene into pET19b, a plasmid containing a sequence encoding a string of ∼10 histidines, and expressing the protein in Escherichia coli BLR(DE3). The tagged YZGD expresses well, is soluble, and retains full activity. It appears to be completely purified by nickel affinity chromatography. This will now allow YZGD to be purified in large quantities, crystallized, and its structure determined.

Indexing (details)


Subject
Biochemistry;
Bioinformatics
Classification
0487: Biochemistry
0715: Bioinformatics
Identifier / keyword
Pure sciences; Biological sciences; Enzymology; HAD superfamily; His-tag; Nudix hydrolase; Protein purification; Pyridoxal phosphate
Title
YZGD pyridoxal phosphatase from Paenibacillus thiaminolyticus; subcloning, expression, and purification for x-ray crystallography structure determination
Author
Strassner, Amanda M.
Number of pages
48
Publication year
2008
Degree date
2008
School code
0465
Source
MAI 46/05M, Masters Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
ISBN
9780549525066
Advisor
O'Handley, Suzanne F.
Committee member
Kim, Thomas D.; Savka, Michael A.
University/institution
Rochester Institute of Technology
Department
Bioinformatics
University location
United States -- New York
Degree
M.S.
Source type
Dissertations & Theses
Language
English
Document type
Dissertation/Thesis
Dissertation/thesis number
1452854
ProQuest document ID
304388125
Copyright
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
http://search.proquest.com/docview/304388125
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