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Inositol polyphosphatases (IPPases) play an important role in the metabolism of inositol polyphosphates, a class of molecules involved in signal transduction. Here we characterize 2 new protein tyrosine phosphatase-like IPPases (PhyAsl and PhyBsl) cloned from Selenomonas lacticifex that can hydrolyze myo-inositol hexakisphosphate (InsP6) in vitro. To determine their preferred substrates and stereospecificity of InsP6 dephosphorylation, a combination of kinetic and high-performance ion pair chromatography studies were conducted. Despite only 33% amino acid sequence identity between them, both enzymes display strict specificity for IPP substrates and cleave InsP6 primarily at the d-3-phosphate position (>90%). Furthermore, both enzymes predominantly degrade InsP6 to Ins(2)P via identical and very specific routes of dephosphorylation (3,4,5,6,1). Despite these similarities, PhylAsl is shown to have a slight kinetic preference for the major inositol pentakisphosphate intermediate in its InsP6 hydrolysis pathway, whereas PhyBsl displays a unique and substantial preference for an inositol tetrakisphosphate intermediate.

Two experiments were conducted with pigs fitted with simple T-cannulas at the distal ileum to evaluate the effect of energy (ME) intake implemented by variation of feeding level (FL) (exp. 1) or dietary plant oil content (exp. 2), on apparent ileal (AID) and total tract digestibilities (ATTD) of phosphorus (P) and calcium (Ca), including measurements of lower myo-inositol phosphates at the ileal and fecal level. In exp. 1, 14 barrows (BW 7.3 kg) were fed one of two low-P corn-soybean meal diets providing either 600 U kg-1 microbial (Natuphos® 5000 G) or native wheat phytase at two FL (2.0 vs. 2.8 × ME maintenance requirement) according to a two-period cross-over design. The AID and ATTD of P and Ca were higher (P < 0.05) in pigs fed microbial phytase compared with wheat phytase. Furthermore, the recoveries of lower myo-inositol phosphates in the feces, but not in the ileal digesta, were higher (P < 0.05) when microbial instead of wheat phytase was added to the diet. Increasing the feed intake improved (P < 0.05) overall AID and ATTD of P, independent of the origin of the supplemental phytase. Additionally, the increase in FL improved (P < 0.05) AID of P in pigs fed wheat phytase. There was no effect (P > 0.05) of FL on ileal or fecal recovery of myo-inositol phosphates. In exp. 2, eight barrows (BW 20.3 kg) were fed a low-P corn-soybean meal diet providing 600 U kg-1 microbial phytase (Natuphos® 5000 G) supplemented without or with 6% plant oil according to a two-period cross-over design. There was no effect (P > 0.05) of oil supplementation on AID and ATTD of P or Ca. In conclusion, an increase in FL improves AID and ATTD of P in pigs fed diets supplemented with microbial or wheat phytase, while increasing energy density had no effect. Key words: Feeding level, phytase, phosphorus, calcium, pigs, digestibility

For the first time a dual pathway for dephosphorylation of myo-inositol hexakisphosphate by a histidine acid phytase was established. The phytate-degrading enzyme of Klebsiella terrigena degrades myo-inositol hexakisphosphate by stepwise dephosphorylation, preferably via D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, D-Ins(1,2)P2 and alternatively via D-Ins(1,2,4,5,6)P5, Ins(2,4,5,6)P4, D-Ins(2,4,5)P3, D-Ins(2,4)P2 to finally Ins(2)P. It was estimated that more than 98% of phytate hydrolysis occurs via D-Ins(1,2,4,5,6)P5. Therefore, the phytate-degrading enzyme from K. terrigena has to be considered a 3-phytase (EC 3.1.3.8). A second dual pathway of minor importance could be proposed that is in accordance with the results obtained by analysis of the dephosphorylation products formed by the action of the phytate-degrading enzyme of K. terrigena on myo-inositol hexakisphosphate. It proceeds preferably via D-Ins(1,2,3,5,6)P5, D-Ins(1,2,3,6)P4, Ins(1,2,3)P3, D-Ins(2,3)P2 and alternatively via D-Ins(1,2,3,5,6)P5, D-Ins(2,3,5,6)P4, D-Ins(2,3,5)P3, D-Ins(2,3)P2 to finally Ins(2)P. D-Ins(2,3,5,6)P4, D-Ins(2,3,5)P3, and D-Ins(2,4)P2 were reported for the first time as intermediates of enzymatic phytate dephosphorylation. A role of the phytate-degrading enzyme from K. terrigena in phytate breakdown could not be ruled out. Because of its cytoplasmatic localization and the suggestions for substrate recognition, D-Ins(1,3,4,5,6)P5 might be the natural substrate of this enzyme and, therefore, may play a role in microbial pathogenesis or cellular myo-inositol phosphate metabolism.Key words: myo-inositol phosphate isomers, phytate-degrading enzyme, phytate, phytase, Klebsiella terrigena.

The pathway of dephosphorylation of myo-inositol hexakisphosphate by the phytate-degrading enzymes of Bacillus subtilis 168, Bacillus amyloliquefaciens ATCC 15841, and Bacillus amyloliquefaciens 45 was established using a combination of high-performance ion chromatography analysis and kinetic studies. The data demonstrate that all the Bacillus phytate-degrading enzymes under investigation dephosphorylate myo-inositol hexakisphosphate by sequential removal of phosphate groups via two independent routes; the routes proceed via D-Ins(1,2,4,5,6)P5 to Ins(2,4,5,6)P4 to finally Ins(2,4,6)P3 or D-Ins(2,5,6)P3 and via D-Ins(1,2,4,5,6)P5 to D-Ins(1,2,5,6)P4 to finally D-Ins(1,2,6)P3. The resulting myo-inositol trisphosphate D-Ins(1,2,6)P3 was degraded via D-Ins(2,6)P2 to finally Ins(2)P after prolonged incubation times in combination with increased enzyme concentration. Key words: Bacillus spp., myo-inositol phosphate isomers, phytase, phytate degradation.