Crystallization and preliminary crystallographic analysis of glyceraldehyde 3-phosphate dehydrogenase from Sacchromyces cerevisiae (baker's yeast)

Rotem Gilboa, Alan Joseph Bauer, Gil Shoham*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

Two related and not thoroughly resolved issues in biochemistry concern the role, if any, of enzyme surfaces in routine metabolism and the method by which metabolic intermediates move between enzyme active sites during multi-step degradation or synthesis. An important enzyme for which a detailed three-dimensional structural analysis has been initiated is yeast glyceraldehyde 3-phosphate dehydrogenase (yGAP-DH). This enzyme is active as a tetramer of total molecular weight of 145 kDa and requires nicotinamide adenine dinucleotide (NAD+) as cofactor. In this report, the crystallization and preliminary crystallographic characterization of several crystal forms of yGAP-DH are described. Of the five distinct crystal forms, the most suitable was found to contain the holo-enzyme, and the crystals were grown by the vapor-diffusion method using polyethylene glycol 6000 as precipitant, sodium acetate as buffer (pH 4.6), and NAD+ and dithiothreitol as additives. The crystals belong to the orthorhombic space group P21212, with cell dimensions of a = 87.33, b = 96.11 and c = 115.34 Å. These crystals are mechanically strong, relatively stable in the X-ray beam and diffract X-rays (from a normal rotating-anode radiation source) to better than 2 Å resolution. A full 2.1 Å resolution diffraction data set (98% completion) has been measured. The three-dimensional structures of related GAP-DH enzymes from several other sources have been determined and reported, and are available for a molecular replacement structure solution.

Original languageAmerican English
Pages (from-to)1467-1470
Number of pages4
JournalActa Crystallographica Section D: Biological Crystallography
Volume54
Issue number6 PART II
DOIs
StatePublished - 1998

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