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 IC50 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.
Arthrobacter sp. EM1 is a cold-adapted bacterium isolated from the Antarctic region, which was known to exhibit mannan-degrading activity. Accordingly, this strain not only promises a cell factory for mannan-degrading enzymes, widely used in industry but also serves as a model organism to decipher its cold adaptation mechanism. Accordingly, whole genome sequencing of the EM1 strain was performed via Single Molecule Real Time sequencing under the PacBio platform, followed by genome HGAP de novo assembly and genome annotation through Rapid Annotation System Technology (RAST) server. The chromosome of this strain is 3,885,750 bp in size with a GC content of 65.8. The annotation predicted a total of 3607 protein-coding genes and 65 RNA genes, which were classified under 398 subsystems. The subsystem with the highest number of genes is carbohydrate metabolism (397 genes), which includes two genes encoding mannan-degrading enzymes (endoglucanase and α-mannosidase). This confirmed that the EM1 strain is able to produce cold-adapted mannan degrading enzymes. The complete genome sequence data have been submitted to the National Center for Biotechnology Information (NCBI) and have been deposited at GenBank (Bioproject ID Accession Number: PRJNA963062; Biosample ID Accession Number: SAMN34434776; GenBank: CP124836.1; https://www.ncbi.nlm.nih.gov/nuccore/CP124836).