Study On Molecular Roots About The Function Of Oxidation For Aflatoxin Oxidase

Aflatoxin oxidase (AFO) has been successfully cloned and expressed in prokaryotic system inour laboratory, previously. It has been found that the enzyme catalyzes the redox reaction of thesubstrate and produce H2O2. Based on the previous findings, in this study, the proofs to supportthe oxidase action of AFO have been investigated by with optimizing expression and purificationconditions of active AFO. This study is significant for further investigation on the relationshipbetween oxidation function and structure of AFO.The full-length amino acid sequences of AFO may match a dipeptidylpeptidaseIII by PBDdatabase with40.4%homologous. However, AFO have been confirmed to be a redoxicase whiledipeptidylpeptidase works as a hydrolase. Thus, the main purpose of this study is to find theredoxicational roots of AFO on molecular level. The main contents of this study include (1)answering whether the AFO redoxication is related to valence metal ion, and what is it;(2)observing whether AFO may combin oxygen, which is meaningful for the suggestion that AFOmight be a kind of monooxygenase;(3) whether the two cysteine of AFO forming anintramolecular disulfide bonds, or existing in the free state which is significant to stabilizing theenzyme or with high possibility involving in the catalytic activity. A high yield, with highaflatoxin oxidase activity was obtained through the optimization of expression conditions.Through the optimization of purification procedures E/AFO with95%purity has been obtained.By using both of DTT, which can open the disulfide bonds, and glutathion, which may react withfree cysteine, it has been confirmed that AFO does not have a disulfide bond, the two cysteinesremain free. By means of isothermal titration calorimetry (ITC) and absorption spectroscopystudies it has been found that there is an effective binding reaction between AFO and copper ions.Inaddition, in the presence of copper ions, AFO demonstrated the ability of oxygen binding.In conclusion: unlike dipeptidyl peptidase, AFO is a redoxidase, and the function ofredoxication for AFO is closely related with the metal ion, copper. Each AFO moleculecombined with one copper ion. The previous findings that AFO may exchange an electronbetween oxidational and reductional state can be explained with the presence of copper ion.