Molecular Regulation Mechanism Underlying The Expression Of L-amina Acid Oxidase In Pseudoalteromonas Sp.RF-1

FAD-binding L-amino acid oxidase(LAAO)catalyzes the oxidative deamination of L-amino acids and thereby produces hydrogen peroxide(H2O2),ammonia(NH4+)and the corresponding a-keto acids.The enzyme exhibits a wide range of biological activities including apoptosis-inducing,edema-inducing,inhibition or induction of platelet aggregation,antibacterial effect and antiviral activity,thus attracting great attention.In this study,we tried to reveal the regulation mechanism underlying the expression of LAAO in Pseudoalteromonas sp.Rf-1.First,a new quantitative in-gel determination of LAAO activity based on ferric-xylenol orange(Fe?XO)formation was established.Due to the conversion of Fe? to Fe? by H2O2 and subsequent formation of Fe?Xo complex halo in agar medium,the logarithm of H2O2 concentration from 5 to 160 ?M was linearly correlated to the diameter of purplish red Fe?XO halo.By extracting the LAAO-generated H2O2 concentration,the LAAO activity can be quantitatively determined.This method is not only highly sensitive to detect H2O2 down to micromolar range,but also easy to handle,cheap,reproducible,convenient and accurate.Second,the suicide plasmid pLOF/Km in donor E.coli S17-1(??),bearing a transposon mini-Tn10,was transferred into the recipient Pseudoalteromonas sp.Rf-1 for the generation of a mutant library through conjugation.More than four thousand individual transposon inserted mutants were gained.Among them,twelve mutants of Rf-1 with deficient or altered LAAO activity were screened out with Fe?XO formation method.Of these 12 mutants,three mutants had the increased LAAO activity,eight mutants had the reduced LAAO activity,and four mutants exhibited the defective LAAO activity.The DNA sequence flanking transposon in the mutant was determined using thermal asymmetric PCR and arbitrary PCR.Most of the inserted sequences could be matched with the genes from P.rubra ATCC 29570,except the mutant B10 whose inserted sequence showed the high identity(68%)to a tbdR gene of Alcanivorax dieselolei B5.The inserted sequence of mutant B22 showed the highest identity(72%)to the gntR gene.The inserted sequences of mutants Bll,B17 and A15 had the highest identities to a hypothetical protein gene(88%),nrpS gene(82%)and upstream non-coding sequences of Methylase gene(80%),respectively.The inserted sequences of mutants B1,B6,B12 and B19 yielded the highest identity to sdmt gene(80%),karo gene(80%),nat5 gene(80%)and nhaD gene(81%),respectively.Finally,genetic complementation was conducted by cloning sdmt and nhaD into plasmid pBBR1MCS-5 to construct the recombinant plasmids pBBR1MCS-5sdmt and pBBR1MCS-5nhaD which were subsequently transferred into mutants B1 and B19,respectively.After complementation,the mutants B1 and B19 were almost totally restored to produce LAAO.These results suggest that LAAO expression in Rf-1 is controlled by sdmt and nhaD genes.