SO-LAAO, a novel L-amino acid oxidase that enables Streptococcus oligofermentans to over-compete Streptococcus mutans by generating H₂O₂ from peptone

State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing100101, China; School of Dentistry, University of California, Los Angeles, CA 90095; Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095; Oral Biology, College of Dentistry and Microbiology and Immunology, College of Medicine University of Oklahoma Health Sciences Center BRC366, Oklahoma City, Ok 73104

^* To whom correspondence should be addressed. Email: dongxz{at}sun.im.ac.cn.

	Abstract

We previously demonstrated that Streptococcus oligofermentans suppressed the growth of Streptococcus mutans, the primary cariogenic pathogen, by producing hydrogen peroxide (H₂O₂) through lactate oxidase (Lox) activity. In this study, we found that the lox mutant of S. oligofermentans regained the inhibition while growing on peptone rich plates. Further studies demonstrated that the H₂O₂ produced on peptone by S. oligofermentans was mainly derived from seven L-amino acids, i.e. L-aspartic acid, L-tryptophan, L-lysine, L-isoleucine, L-arginine, L-asparagine and L-glutamine, indicating the possible existence of L-amino acids oxidase (L-AAO) that can produce H₂O₂ from L-amino acids. Through searching S. oligofermentans genome for ORFs with a conserved FAD binding motif that exists in the known L-AAOs including those of snake venom, fungi and bacteria, a putative L-amino acids oxidase gene, assigned as aao_so, was cloned and over expressed in Escherichia coli. The purified protein SO-LAAO showed a molecular weight of 43 KDa on SDS-PAGE, and catalyzed H₂O₂ formation from the seven L-amino acids determined above, thus confirming its L-amino acid oxidase activity. The SO-LAAO identified in S. oligofermentans differed evidently from the known L-AAOs in both substrate profile and sequence, suggesting that it could represent a novel L-amino acid oxidase. An aao_so mutant of S. oligofermentans did lose H₂O₂ formation from the seven L-amino acids, further verifying its function as an L-AAO. Furthermore the inhibition of S. oligofermentans over S. mutans in a peptone rich mix-species biofilm was greatly reduced in the aao_so mutant, indicating its importance in interspecies competition.