科研之友
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摘要
The BT4131 gene from the bacterium Bacteroides thetaiotaomicron VPI-5482 has been cloned and overexpressed in Escherichia coli. The protein, a member of the haloalkanoate dehalogenase superfamily (subfamily 1113), was purified to homogeneity, and its X-ray crystal structure was determined to 1.9 angstrom resolution using the molecular replacement phasing method. BT4131 was shown by an extensive substrate screen to be a broad-range sugar phosphate phosphatase. On the basis of substrate specificity and gene context, the physiological function of BT4131 in chitin metabolism has been tentatively assigned. Comparison of the BT4131 structure alpha/beta cap domain structure with those of other type 1113 enzymes (phosphoglycolate phosphatase, trehalose-6-phosphate phosphatase, and proteins of unknown function known as PDB entries 1NF2, 1NRW, and 1RKQ) identified two conserved loops (BT4131 residues 172-182 and 118-130) in the alpha beta beta(alpha beta alpha beta)alpha beta beta type caps and one conserved loop in the alpha beta beta alpha beta beta type caps, which contribute residues for contact with the substrate leaving group. In BT4131, the two loops contribute one polar and two nonpolar residues to encase the displaced sugar. This finding is consistent with the lax specificity BT4131 has for the ring size and stereochemistry of the sugar phosphate. In contrast, substrate docking showed that the high-specificity phosphoglycolate phosphatase (PDB entry IL6R) uses a single substrate specificity loop to position three polar residues for interaction with the glycolate leaving group. We show how active site "solvent cages" derived from analysis of the structures of the type 1113 HAD phosphatases could be used in conjunction with the identity of the residues stationed along the cap domain substrate specificity loops, as a means of substrate identification.
- 出版日期2005-6-21
