Characterization And Structure-function Studies Of Phytases From Yersinia Spp.

Phytate is the primary storage form of phosphorus in cereals and legumes. Phytases (myo-inositolhexakisphosphate phosphohydrolases) hydrolyze the phosphoester bonds of phytate to yield inorganicphosphate and less-substituted inositol. Monogastric animal feed supplemented with microbial phytaseseffectively improves phytate phosphorus utilization, reduces the excretion of phosphorus in animalmanure and improves the nutrient value by removing the anti-nutrient factor of phytate in intensivelivestock production. The industrial demand for phytase with high specific activity, excellentthermostability and acid stability continues to stimulate the search for new enzyme sources and thestudies on the catalytic mechanisms and protein-engineering of phytases.HAP phytases (EC 3.1.3.8) are a class of phytases with excellent catalysis capacity and have beenwidely used in animal feed. Yersinia spp. are excellent microbial sources of HAP phytases,and ten HAPphytase genes have been cloned from 11 species genomes of the genus Yersinia. Among them, HAPphytases from Yersinia intermedia and Y. rohdei had excellent comprehensive properties and applicationpotential. In this study, we heterologously expressed and characterized the genes Ykappa, Yfappa andYeappa from Yersinia kristeensenii, Y. frederiksenii and Y. enterocolitica, respectively. Furthermore, thestudies on catalytic mechanisms through site-directed mutagenesis were conducted.The gene Ykappa from Y. kristeensenii was expressed in Pichia pastoris. The purified recombinantphytase, YkAppa, had optimal activity at 55℃and pH 4.5, exhibited enzymatic activity between pH 2.0and 6.5, with a specific activity of 2,656 U/mg at pH 4.5 and 37℃. YkAppa was highly pH stable,retaining more than 90% of its initial activity after pre-incubation under varying pH conditions (pH1.5–11.0) at 37℃for 3 h. YkAppa was thermostable, and retained 46% of its initial activity afterincubation at 80℃for 10 min. YkAppa also showed efficiency in hydrolysis of phytate phosphorusfrom soybean meal in vitro. Compared with other well-known phytases, YkAppa has excellentcomprehensive properties, such as high specific activity, good pH stability and thermostability, highdegradation efficacy of soybean meal phytate and so on, and has significant potential in feed industryuse.The purified YfAppa, heterologously expressed in Escherichia coli, had optimal activity at pH2.5—substantially lower than that of most of microbial phytases (pH optima 4.5–6.0). The amino acidsequence of YfAppa has the highest identity (84%) to that of Y. intermedia HAP phytase (optimal pH4.5). Based on sequence alignment and molecular modeling of YfAppa and related phytases, only onedivergent residue in YfAppa, Ser51, was identified to be in close proximity to the catalytic site. Tounderstand the acidic adaptation of YfAppa, five mutants (S51A, S51T, S51D, S51K and S51I) wereconstructed by site-directed mutagenesis, expressed in E. coli, purified, and characterized. Ser, Thr andIle are all uncharged amino acids, but replacing Ser with Thr and Ile changed the pH optima a lot.Mutants S51T and S51I exhibited a shift in the optimal pH from 2.5 to 4.5 and 5.0, respectively,confirming the role of Ser51 in defining the optimal pH. Thus, a previously unrecognized factor other than electrostatics—presumably the side-chain structure near the active site—contributes to the optimalpH for YfAppa activity. Compared with wild-type YfAppa, mutant S51T showed higher specificactivity, greater activity over pH 2.0–5.5, and increased thermal and acid stability. These propertiesmake mutant S51T a better candidate than the wild-type YfAppa for use in animal feed.The maximum activity of YeAppa occurs at pH 5.0 and 45℃, and notably its specific activityunder physiological conditions (3.28 U/mg, pH 5.0 and 37℃) is 800-fold less than that of its Y.kristeensenii homolog (YkAppa; 88% amino acid sequence identity). Sequence alignment andmolecular modeling showed that the arginine at position 79 (Arg79) in YeAppa corresponding to Gly inYkAppa as well as other HAP phytases is the only non-conserved residue near the catalytic site.Site-directed replacement of Arg79 with Gly increased the specific activity of YeAppa to 2,615 U/mg,indicating that Arg79 is primarily responsible for the decreased activity. To characterize the effects ofother residues at this position on the specific activities of phytases, E. coli EcAppa, a well-characterizedphytase with a known crystal structure, was selected for mutagenesis—its Gly73 was replaced with Arg,Asp, Glu, Ser, Thr, Leu, or Tyr. The specific activities of all of the corresponding EcAppa mutants wereless than that of the wild-type phytase, and the activity levels were approximately proportional to themolecular volumes of the substituted residues'side chains. Thus, a new determinant that influences thecatalytic efficiency of HAP phytases—the molecular volumes of the substituted residues'side chainshas been identified.In summary, phytases from Yersinia spp. are highly identical in amino acid sequences but varied inproperties, and are good materials for structure-function studies. This study obtained a phytase YkAppawith excellent comprehensive properties and important application potential, identified two factorscontributing to pH optima and catalytic efficiency of HAP phytases, and thus had great significance intheory and application.