Biochemical and molecular characterization of a novel type of Mutanase from Paenibacillus sp. strain RM1: identification of its mutan-binding domain, essential for degradation of Streptococcus mutans biofilms.
A novel type of mutanase (termed mutanase RM1) was isolated from Paenibacillus sp. strain RM1. The purified enzyme specifically hydrolyzed alpha-1,3-glucan (mutan) and effectively degraded biofilms formed by Streptococcus mutans, a major etiologic agent in the progression of dental caries, even following brief incubation. The nucleotide sequence of the gene for this protein contains a 3,873-bp open reading frame encoding 1,291 amino acids with a calculated molecular mass of 135 kDa. The protein contains two major domains, the N-terminal domain (277 residues) and the C-terminal domain (937 residues), separated by a characteristic sequence composed of proline and threonine repeats. The characterization of the recombinant proteins for each domain which were expressed in Escherichia coli demonstrated that the N-terminal domain had strong mutan-binding activity but no mutanase activity whereas the C-terminal domain was responsible for mutanase activity but had mutan-binding activity significantly lower than that of the intact protein. Importantly, the biofilm-degrading activity observed with the intact protein was not exhibited by either domain alone or in combination with the other. Therefore, these results indicate that the structural integrity of mutanase RM1 containing the N-terminal mutan-binding domain is required for the biofilm-degrading activity.
Shimotsuura
Isao
I
Oral Care Research Laboratories, Lion Corporation, 3-7 Honjyo 1-chome, Sumida-ku, Tokyo 130-8644, Japan. isaosimo@lion.co.jp
Kigawa
Hiromitsu
H
Ohdera
Motoyasu
M
Kuramitsu
Howard K
HK
Nakashima
Syozi
S
D000595:Amino Acid Sequence; D001665:Binding Sites; D018441:Biofilms / Q000187:drug effects / Q000254:growth & development; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005936:Glucans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006094:Gram-Positive Bacteria / Q000201:enzymology* / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D017422:Sequence Analysis, DNA; D013295:Streptococcus mutans / Q000187:drug effects / Q000254:growth & development; D013696:Temperature
D004269:DNA, Bacterial; D005936:Glucans; D011994:Recombinant Proteins; C045788:alpha-1,3-glucan; D006026:Glycoside Hydrolases; C021780:exo-1,3-alpha-glucanase