The hyperthermophilic bacterium encodes a gene sharing sequence similarities with several known genes for alkaline phosphatase (AP). common divalent metal ion necessary for activity in known APs, was proven to inhibit the phosphatase enzyme at concentrations above 0.3 moles Zn: 1 mole monomer. All activity was abolished in the current presence of 0.1 mM EDTA. The AP principal sequence is 28% identical in comparison to AP. Predicated on a structural model, the energetic sites are superimposable aside from two residues close to the AP Mg binding site, D153 and K328 (numbering) corresponding to histidine and tryptophan in AP, respectively. Sucrose-density gradient sedimentation experiments demonstrated that the proteins exists in a number of quaternary forms predominated by an octamer. and AP is normally a well-characterized homodimeric metalloenzyme, it could serve as a basis for evaluation to enzymes with comparable homology. Proteins from extremophilic organisms are of interest for his or her unique characteristics and stability. isolated from geothermal heated marine sediment, represents the first hyperthermophile found from bacterial, not archaeal, origins (Huber et al. 1986). Its genome is the result of lateral gene transfer because 52% of predicted coding sequences are most similar to proteins in bacterial species, namely, grows optimally at a temp of 80C and a salt concentration of 2.5 M. It is gram-bad and has a unique outer sheath structure (Huber et al. 1986). From sequence alignment, the AP gene is closely related to the gene, which generates a homodimeric AP requiring Co(II) for activity. AP also shows strong homology with the gene product, the monomeric Co(II)-requiring AP, and the 537705-08-1 enzyme. We have determined the activity, quaternary structure, thermostability, and metallic preference of AP. The preference for Co(II) in AP is definitely analogous to the metallic dependence found in the enzymes. On assessment with the Zn(II)-requiring AP, it can be shown that this change in metallic preference may be attributed to specific amino acid substitutions near the metal-binding positions in the active site. Results and Conversation Alignment, sequence assessment, and modeling A protein BLAST search was performed to find sequences that most closely matched the AP sequence (Table 1?1).). The gene products from and genes were among the most homologous proteins. They were chosen for further study because the signal sequence cleavage point and metallic requirement for these enzymes experienced already been characterized (Hulett et al. 1990; Hulett et al. 1991). Although the AP was not among the most similar, it is the only AP from a bacterial resource whose structure offers been solved (Kim and Wyckoff 1991); consequently, it 537705-08-1 was also used for comparisons to AP. Table 1. Closest matches to the T. maritima AP sequence 537705-08-1 from the BLAST databasea APMetals153328ReferenceAP metal-binding and active-site residues are indicated. b NCBI database accession quantity. A main sequence alignment was performed using the AP sequence and three homologous AP enzymes (Fig. 1 ?). As seen from the alignment, AP is very similar to both the AP proteins and the well-characterized AP, especially in the active site and metal-binding regions. This sequence alignment was also used to generate a structural model of the monomeric AP using the structure 1ED9 (Stec et al. 2000) with the program SWISS-MODEL (Peitsch 1995; Guex et al. 1999; Guex and Peitsch 1997). The sequence alignment and structural model were used to compare the active sites of and AP (Fig. 2A ?), along with the overall predicted structure Rabbit polyclonal to smad7 of 537705-08-1 the monomer (Fig. 2B ?). Open in a separate window Fig. 1. Sequence alignment of gene products, alkaline phosphatases (APs). Residues associated with metallic binding in the AP active sites are labeled with (). These residues are completely conserved in all four of these examples of AP, with the exception of D153H and K328W (mature AP numbering). The N-terminus of the AP mature protein is definitely indicated with (?),.