TY - JOUR

T1 - Raffinose family oligosaccharide utilisation by probiotic bacteria: insight into substrate recognition, molecular architecture and diversity of GH36 alpha-galactosidases

A1 - Abou Hachem,Maher

A1 - Fredslund,Folmer

A1 - Andersen,Joakim Mark

A1 - Larsen,Rene Jonsgaard

A1 - Majumder,Avishek

A1 - Hansen,Morten Ejby

A1 - van Zanten,Gabriella Christina

A1 - Lahtinen,S. J.

A1 - Barrangou,R.

A1 - Klaenhammer,T.

A1 - Jacobsen,S.

A1 - Coutinho,P. M.

A1 - Lo Leggio,Leila

A1 - Svensson,Birte

AU - Abou Hachem,Maher

AU - Fredslund,Folmer

AU - Andersen,Joakim Mark

AU - Larsen,Rene Jonsgaard

AU - Majumder,Avishek

AU - Hansen,Morten Ejby

AU - van Zanten,Gabriella Christina

AU - Lahtinen,S. J.

AU - Barrangou,R.

AU - Klaenhammer,T.

AU - Jacobsen,S.

AU - Coutinho,P. M.

AU - Lo Leggio,Leila

AU - Svensson,Birte

PB - Informa Healthcare

PY - 2012

Y1 - 2012

N2 - The organisation of genes conferring utilisation of raffinose family oligosaccharides (RFOs) has been analysed in several probiotic bacteria from the Bifidobacterium and Lactobacillus genera. Glycoside hydrolase family 36 (GH36) alpha-galatosidase encoding genes occur together with sugar transport systems of the glycoside-pentoside-hexuronide cation symporter family (GPH), sugar phosphotransferase systems (PTSs) or ATP-binding cassette systems (ABCs) highlighting the diversity of RFO uptake. The GH36 genes are often clustered together with sucrose hydrolases or phosphorylases ensuring the degradation of RFO to monosaccharides. Differential proteomics and transcriptomics data from our laboratories implicated ABC transporters in the uptake of RFO in both Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bl-04. Interestingly, only one of three GH36 encoding genes in B. animalis subsp. lactis Bl-04 was upregulated upon growth on RFO, suggesting that the other two gene products may have different specificities. The structure of the GH36 homotetrameric alpha-galactosidase from L. acidophilus NCFM (LaMel36A) was determined in complex with galactose bound in the active site to 1.58 angstrom. Differences in the N- and C-terminal domains of the LaMel36A monomer distinguished it from the monomeric TmGalA from Thermotoga maritima providing a structural rationale for the observed difference in oligomeric states of the two enzymes. Tetramerisation of LaMel36A creates a narrow and deep active site pocket between three monomers, which explains the preference of tetrameric GH36 enzymes for RFO and their lack of activity on polymeric galacto(gluco) mannan. Finally, GH36 was divided into four subgroups based on active site motifs, which illuminates functional and structural diversity in the family and aids further annotation of emerging sequences.

AB - The organisation of genes conferring utilisation of raffinose family oligosaccharides (RFOs) has been analysed in several probiotic bacteria from the Bifidobacterium and Lactobacillus genera. Glycoside hydrolase family 36 (GH36) alpha-galatosidase encoding genes occur together with sugar transport systems of the glycoside-pentoside-hexuronide cation symporter family (GPH), sugar phosphotransferase systems (PTSs) or ATP-binding cassette systems (ABCs) highlighting the diversity of RFO uptake. The GH36 genes are often clustered together with sucrose hydrolases or phosphorylases ensuring the degradation of RFO to monosaccharides. Differential proteomics and transcriptomics data from our laboratories implicated ABC transporters in the uptake of RFO in both Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bl-04. Interestingly, only one of three GH36 encoding genes in B. animalis subsp. lactis Bl-04 was upregulated upon growth on RFO, suggesting that the other two gene products may have different specificities. The structure of the GH36 homotetrameric alpha-galactosidase from L. acidophilus NCFM (LaMel36A) was determined in complex with galactose bound in the active site to 1.58 angstrom. Differences in the N- and C-terminal domains of the LaMel36A monomer distinguished it from the monomeric TmGalA from Thermotoga maritima providing a structural rationale for the observed difference in oligomeric states of the two enzymes. Tetramerisation of LaMel36A creates a narrow and deep active site pocket between three monomers, which explains the preference of tetrameric GH36 enzymes for RFO and their lack of activity on polymeric galacto(gluco) mannan. Finally, GH36 was divided into four subgroups based on active site motifs, which illuminates functional and structural diversity in the family and aids further annotation of emerging sequences.

KW - functional diversity

KW - GenBank sequence data

KW - molecular architecture

KW - structural diversity

KW - substrate recognition

KW - Actinomycetes and Related Organisms Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Irregular Nonsporing Gram-Positive Rods [08890] Bifidobacterium animalis lactis subspecies strain-Bl-04

KW - Anaerobic Gram-Negative Rods Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Bacteroidaceae [06901] Thermotoga maritima species

KW - Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Regular Nonsporing Gram-Positive Rods [07830] Lactobacillus acidophilus species strain-NCFM

KW - Facultatively Anaerobic Gram-Negative Rods Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Enterobacteriaceae [06702] Escherichia coli species

KW - alpha-galatosidase

KW - galactomannan 11078-30-1

KW - galactose 26566-61-0

KW - genes

KW - glycoside hydrolase family 36 GH36

KW - Mel36A monomer N-terminal domain, C-terminal domain

KW - monosaccharides

KW - phosphorylase 9035-74-9 EC 2.4.1.1

KW - probiotic

KW - raffinose family oligosaccharides RFO

KW - sucrose hydrolases

KW - 03502, Genetics - General

KW - 10062, Biochemistry studies - Nucleic acids, purines and pyrimidines

KW - 10068, Biochemistry studies - Carbohydrates

KW - 10802, Enzymes - General and comparative studies: coenzymes

KW - 31000, Physiology and biochemistry of bacteria

KW - 31500, Genetics of bacteria and viruses

KW - Biochemistry and Molecular Biophysics

KW - Enzymology

KW - Molecular Genetics

U2 - 10.3109/10242422.2012.674717

DO - 10.3109/10242422.2012.674717

JO - Biocatalysis and Biotransformation

JF - Biocatalysis and Biotransformation

SN - 1024-2422

IS - 3

VL - 30

SP - 316

EP - 325

ER -