Identification and characterization of an extracellular alkaline phosphatase in the marine diatom Phaeodactylum tricornutum.
In phosphorus-deficient conditions, Phaeodactylum tricornutum releases an alkaline phosphatase (PtAPase) to the medium that is readily detectable by activity staining. Nucleic acid and amino acid sequence of this alkaline phosphatase (APase) was identified by performing proteomic analysis and database searches. Sequence alignment suggests that PtAPase belongs to the PhoA family, and it possesses key residues at the Escherichia coli PhoA active site. Quantitative PCR results indicate that the induction of APase mRNA transcription is very sensitive to phosphorus availability and population growth. The molecular mass of native PtAPase (148 kDa) determined by gel filtration chromatography indicates that PtAPase, like most PhoA, is homodimeric. Zn and Mg ions are essential cofactors for most PhoA enzymes; however, PtAPase activity did not require Zn ions. In fact, 5 mM Zn²⁺, Mo²⁺, Co²⁺, Cd²⁺, or Cu²⁺ inhibited its enzymatic activity, whereas 5 mM Mn²⁺, Mg²⁺, or Ca²⁺ enhanced its enzymatic activity. The responses of PtAPase to divalent metal ions were different from those of most PhoAs, but were similar to the PhoA in a marine bacterium, Cobetia marina. Phylogenetic analysis shows that homologs of PhoA are also present in other diatom species, and that they clustered in a unique branch away from other PhoA members. PtAPase may represent a novel class of PhoA that helps diatoms to survive in the ocean. Quantification of the PtAPase mRNA may help monitor the physiological condition of diatoms in natural environments and artificial bioreactors.
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung 20224, Taiwan.
Hung-Yun
HY
Lin
Chi-Yu
CY
Shih
Hung-Chun
HC
Liu
Jeng
J
Chang
Ying-Lan
YL
Chen
Yet-Ran
YR
Chen
Han-Tso
HT
Lin
Yu-Yung
YY
Chang
Chun-Hua
CH
Hsu
Han-Jia
HJ
Lin
D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D016000:Cluster Analysis; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D017377:Diatoms / Q000201:enzymology*; D019281:Dimerization; D045604:Extracellular Fluid / Q000201:enzymology*; D005786:Gene Expression Regulation / Q000502:physiology*; D019216:Metals, Heavy / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010758:Phosphorus / Q000032:analysis; D010802:Phylogeny; D011157:Population Dynamics; D040901:Proteomics; D016415:Sequence Alignment; D013045:Species Specificity
D017931:DNA Primers; D019216:Metals, Heavy; D010758:Phosphorus; D000469:Alkaline Phosphatase