SIB1 FKBP22 is a peptidyl prolyl cis-trans isomerase (PPIase) member from a psychrotrophic bacterium, Shewanella sp. SIB1, consisting of N- and C-domains responsible for dimerization and catalytic PPIase activity, respectively. This protein was assumed to be involved in cold adaptation of SIB1 cells through its dual activity of PPIase activity and chaperone like-function. Nevertheless, the catalytic inhibition by FK506 and its substrate specificity remain unknown. Besides, ability of SIB1 FKBP22 to inhibit phosphatase activity of calcinuerin is also interesting to be studied since it may reflect wider cellular functions of SIB1 FKBP22. In this study, we found that wild type (WT) SIB1 FKBP22 bound to FK506 with IC of 77.55 nM. This value is comparable to that of monomeric mutants (NNC-FKBP22, C-domain+ and V37R/L41R mutants), yet significantly higher than that of active site mutant (R142A). In addition, WT SIB1 FKBP22 and monomeric variants were found to prefer hydrophobic residues preceding proline. Meanwhile, R142A mutant has wider preferences on bulkier hydrophobic residues due to increasing hydrophobicity and binding pocket space. Surprisingly, in the absence of FK506, SIB1 FKBP22 and its variants inhibited, with the exception of N-domain, calcineurin phosphatase activity, albeit low. The inhibition of SIB1 FKBP22 by FK506 is dramatically increased in the presence of FK506. Altogether, we proposed that local structure at substrate binding pocket of C-domain plays crucial role for the binding of FK506 and peptide substrate preferences. In addition, C-domain is essential for inhibition, while dimerization state is important for optimum inhibition through efficient binding to calcineurin.
While phosphorus (P) is a vital element for the plant to grow, only 0.1% of the phosphate soil is directly to be uptake by plants. Consequently, P fertilizer, which is mostly taken from unrenewable resources of phosphate rock, is practically added into the croplands. Nevertheless, as the demand for this fertilizer kept increasing, the availability of resources and environmental issues are currently raising wide concerns. Alternatively, soil phosphate solubilizing bacteria (PSB) is promising to be further developed as a biofertilizer to increase the availability of P elements for plants. This study aims to screen and characterize novel PSB from the tropical rainforest soil. The soil samples were collected from the Danum Valley tropical rainforest, Sabah. Phosphatase solubilizing bacteria were then screened using the NBRIP Agar selective media. The screening results yielded five colonies, designated as PSB1, PSB2, PSB3, PSB4, and PSB5, displaying halos, with an average diameter of 10mm. Further, 16s rRNA gene sequence analysis using BLASTn suggested that PSB1, PSB2, PSB3, PSB4, and PSB5 were designated as Bacillus sp. PSB01, Pseudomonas oryzyhabitans PSB02, Staphylococcus pasteuri PSB03, Paenibacillus sp. PSB04, and Staphylococcus pasteuri PSB05, respectively. Interestingly, the Paenibacillus group is a promising biofertilizer and is currently used in the global agriculture industry. Accordingly, Paenibacillus sp. PSB04 was then selected for further characterization using Gram staining and observed under scanning electron microscope (SEM). The Gram staining revealed that Paenibacillus sp. PSB04 is a Gram-negative bacterium with a rod shape, which is in good agreement with the SEM result. The specific phosphatase activity of the extracellular fraction of this bacterium was 7378.12 U mg-1 which is the highest activity compared to previous studies. This study provides an early insight into an excellent phosphate solubilizing bacterium for the agriculture industry obtained from Danum Valley.
A Phosphate-Solubilising Bacterium (PSB) of Paenibacillus sp. PSB04 was previously isolated from the Sabah tropical rainforest in Malaysia. Interestingly, the genome sequence of the PSB04 strain harbored an Alkaline Phosphatase (AP) (EC 3.1.3.1) gene and was hypothesized to have unique structural characteristics. Therefore, this study aims to determine the AP three-Dimensional (3D) model and catalytic mechanism from Paenibacillus sp. PSB04 (PAP). To address this, the 3D model of this protein was built and docked into a model substrate of p-nitrophenyl phosphate. As a result, the best complex was shown to have the lowest binding energy of -5.9 kcal/moL. Furthermore, the complex showed the atomic coordination of catalytic residues of PAP and the substrate was similar to that of AP from Escherichia coli (ECAP), which implies that both APs shared a similar catalytic mechanism. In this mechanism, Ser 94 of PAP acted as nucleophilic residues, which were activated by the Zn ion. Arg 145 is predicted to be mobile due to its location in the loop region, which allows this residue to stabilize the substrate through direction or watermediated secondary interaction. Docking simulation of pNPP indicated that the putative residues involved in the catalysis mainly are Ser 94, Ser 141, Ala 146, Thr 147, Pro 148, Asp 293, and Glu 294. Glu 294 is considered a unique residue corresponding to Lys 328 ECAP, allowing the PAP to have a better affinity to stabilize the substrate in the binding cavity. Accordingly, a unique catalytic mechanism of PAP was proposed.
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