Molecular properties of the glucosaminidase AcmA from Lactococcus lactis MG1363: mutational and biochemical analyses.
The major autolysin AcmA of Lactococcus lactis ssp. cremoris MG1363 is a modular protein consisting of an N-terminal signal sequence, a central enzymatic region (glu(acma) as a glucosaminidase), and a C-terminal cell-recognition domain (LysM123). glu(acma) (about 160 amino acids) belongs to the glycoside hydrolase (GH) 73 family, and the two acidic residues E128 and D153 have been thought to be catalytically important. In this study, amino-acid substitution analysis of AcmA was first carried out in the Escherichia coli system. Point mutations E94A, E94Q, E128A, D153A, and Y191A markedly reduced cell-lytic activity (3.8%, 1.1%, 4.2%, 4.8%, and 2.4%, respectively), whereas E128Q and D153N retained significant residual activities (32.1% and 44.0%, respectively). On the other hand, Y191F and Y191W mutations retained high activities (66.2% and 46.0%, respectively). These results showed that E94 (rather than E128 and D153) and the aromatic residue Y191 probably play important roles in catalysis of AcmA. Together with mutational analysis of another GH73 glucoaminidase Glu(atlwm) from the Staphylococcus warneri M autolysin Atl(WM), these results suggested that the GH73 members cleave a glycosidic bond via a substrate-assisted mechanism, as postulated in the GH20 members. AcmA and Glu(atlwm) were purified from E. coli recombinant cells, and their enzymatic properties were studied.
Inagaki
Nobuya
N
Molecular Biology Group, Graduate School of Science and Engineering, University of Toyama, Gofuku, Toyama, Japan.
Iguchi
Akinori
A
Yokoyama
Takahiro
T
Yokoi
Ken-ji
KJ
Ono
Yasushi
Y
Yamakawa
Ayanori
A
Taketo
Akira
A
Kodaira
Ken-Ichi
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015345:Oligonucleotide Probes / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; C092497:AcmA protein, Lactococcus lactis; D009113:Muramidase