SO-LAAO, a Novel l -Amino Acid Oxidase That Enables Streptococcus oligofermentans To Outcompete Streptococcus mutans by Generating H 2 O 2 from Peptone

Abstract

ABSTRACT We previously demonstrated that Streptococcus oligofermentans suppressed the growth of Streptococcus mutans , the primary cariogenic pathogen, by producing hydrogen peroxide (H 2 O 2 ) through lactate oxidase 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 H 2 O 2 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 acid oxidase (LAAO) that can produce H 2 O 2 from l -amino acids. Through searching the S. oligofermentans genome for open reading frames with a conserved flavin adenine dinucleotide binding motif that exists in the known LAAOs, including those of snake venom, fungi, and bacteria, a putative LAAO gene, assigned as aao S o , was cloned and overexpressed in Escherichia coli . The purified protein, SO-LAAO, showed a molecular mass of 43 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and catalyzed H 2 O 2 formation from the seven l -amino acids determined above, thus confirming its LAAO activity. The SO-LAAO identified in S. oligofermentans differed evidently from the known LAAOs in both substrate profile and sequence, suggesting that it could represent a novel LAAO. An aao S o mutant of S. oligofermentans did lose H 2 O 2 formation from the seven l -amino acids, further verifying its function as an LAAO. Furthermore, the inhibition by S. oligofermentans of S. mutans in a peptone-rich mixed-species biofilm was greatly reduced for the aao S o mutant, indicating the gene's importance in interspecies competition.