The 1.9 A crystal structure of heat-labile shrimp alkaline phosphatase.
Alkaline phosphatases are non-specific phosphomonoesterases that are distributed widely in species ranging from bacteria to man. This study has concentrated on the tissue-nonspecific alkaline phosphatase from arctic shrimps (shrimp alkaline phosphatase, SAP). Originating from a cold-active species, SAP is thermolabile and is used widely in vitro, e.g. to dephosphorylate DNA or dNTPs, since it can be inactivated by a short rise in temperature. Since alkaline phosphatases are zinc-containing enzymes, a multiwavelength anomalous dispersion (MAD) experiment was performed on the zinc K edge, which led to the determination of the structure to a resolution of 1.9 A. Anomalous data clearly showed the presence of a zinc triad in the active site, whereas alkaline phosphatases usually contain two zinc and one magnesium ion per monomer. SAP shares the core, an extended beta-sheet flanked by alpha-helices, and a metal triad with the currently known alkaline phosphatase structures (Escherichia coli structures and a human placental structure). Although SAP lacks some features specific for the mammalian enzyme, their backbones are very similar and may therefore be typical for other higher organisms. Furthermore, SAP possesses a striking feature that the other structures lack: surface potential representations show that the enzyme's net charge of -80 is distributed such that the surface is predominantly negatively charged, except for the positively charged active site. The negatively charged substrate must therefore be directed strongly towards the active site. It is generally accepted that optimization of the electrostatics is one of the characteristics related to cold-adaptation. SAP demonstrates this principle very clearly.
de Backer
Maaike
M
European Synchrotron Radiation Facility, Grenoble, France.
McSweeney
Sean
S
Rasmussen
Hanne B
HB
Riise
Bjørn W
BW
Lindley
Peter
P
Hough
Edward
E
D000469:Alkaline Phosphatase / Q000737:chemistry*; D000818:Animals; D018360:Crystallography, X-Ray; D033364:Decapoda / Q000737:chemistry / Q000201:enzymology*; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D013696:Temperature
D000469:Alkaline Phosphatase