Any feedback?
Information on EC 2.4.1.345 - phosphatidyl-myo-inositol alpha-mannosyltransferase and Organism(s) Mycolicibacterium smegmatis and UniProt Accession A0QWG6
for references in articles please use BRENDA:EC2.4.1.345Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
EC Tree
2.4.1.345 phosphatidyl-myo-inositol alpha-mannosyltransferaseIUBMB Comments
Requires Mg2+. The enzyme, found in Corynebacteriales, is involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs).
Specify your search results
Word Map
- 2.4.1.345
- mycobacteria
- mannosides
- glycosyltransferase
- envelope
- gdp-man
-
lipomannan
- tuberculosis
- smegmatis
-
mannosylation
- lipoarabinomannan
-
host-pathogen
- glycans
- phospholipids
-
molten
-
lipoglycans
-
globule
-
non-substrate
- medicine
- analysis
The taxonomic range for the selected organisms is: Mycolicibacterium smegmatis
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Reaction Schemes
+
=
+
+
=
+
Synonyms
phosphatidyl-myo-inositol mannosyltransferase a, mannosyltransferase pima, phosphatidyl-myo-inositol mannosyltransferase pima, phosphatidylinositol mannosyltransferase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
alpha-D-mannose-alpha(1,2)-phosphatidyl-myoinositol transferase
-
-
mannosyltransferase PimA
-
-
-
-
PimA
PimA
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
transfers one or more alpha-D-mannose residues from GDP-mannose to positions 2,6 and others in 1-phosphatidyl-myo-inositol
based on structural, calorimetric, and mutagenesis studies, a model is proposed wherein PimA attaches to the membrane through its N-terminal domain, and this association leads to enzyme activation
SYSTEMATIC NAME
IUBMB Comments
GDP-alpha-D-mannose:1-phosphatidyl-1D-myo-inositol 2-alpha-D-mannosyltransferase (configuration-retaining)
Requires Mg2+. The enzyme, found in Corynebacteriales, is involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs).
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
GDP-alpha-D-mannose + 1-phosphatidyl-1D-myo-inositol
GDP + 2-O-(alpha-D-mannosyl)-1-phosphatidyl-1D-myo-inositol
-
-
-
?
GDP-mannose + 1,2-dioctanoyl-sn-glycero-3-phosphoinositol
?
-
-
-
?
GDP-mannose + phosphatidylinositol
GDP + phosphatidyl-(2-O-alpha-D-manno-pyranosyl)-myo-inositol
GDP-mannose + phosphatidylinositol
GDP + phosphatidyl-(2-O-alpha-D-mannopyranosyl)-myo-inositol
-
-
-
?
GDP-mannose + phosphatidylinositol
GDP + phosphatidyl-(2-O-alpha-D-manno-pyranosyl)-myo-inositol
-
-
-
?
GDP-mannose + phosphatidylinositol
GDP + phosphatidyl-(2-O-alpha-D-manno-pyranosyl)-myo-inositol
-
-
-
?
GDP-mannose + phosphatidylinositol
GDP + phosphatidyl-(2-O-alpha-D-manno-pyranosyl)-myo-inositol
involved in the early steps of phosphatidylinositol mannoside synthesis, the synthesis of phosphatidylinositol monomannosides and derived higher phosphatidylinositol mannosides in M. smegmatis appears to be dependent on PimA and essential for growth
-
-
?
GDP-mannose + phosphatidylinositol
GDP + phosphatidyl-(2-O-alpha-D-manno-pyranosyl)-myo-inositol
PimA is responsible for the initial mannosylation of phosphatidylinositol
-
-
?
additional information
?
-
-
the PimA enzyme catalyzes the transfer of the first Man residue from GDP-Man to the myo-inositol residue of phosphatidylinositol, yielding diacylated forms of phosphatidylinositol mono-mannoside
-
-
?
additional information
?
-
the PimA enzyme catalyzes the transfer of the first Man residue from GDP-Man to the myo-inositol residue of phosphatidylinositol, yielding diacylated forms of phosphatidylinositol mono-mannoside
-
-
?
additional information
?
-
-
essential enzyme for mycobacterial growth
-
-
?
additional information
?
-
essential enzyme for mycobacterial growth
-
-
?
additional information
?
-
-
neither methyl 6-deoxy-6-[(2R)-2,3-dipalmitoyloxypropyloxy]-hydroxyphosphinyl-beta-D-galactopyranoside nor methyl 6-deoxy-6-dihydroxyphosphinyl-beta-D-galactopyranoside serve as substrate for PimA
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
GDP-mannose + phosphatidylinositol
GDP + phosphatidyl-(2-O-alpha-D-manno-pyranosyl)-myo-inositol
GDP-mannose + phosphatidylinositol
GDP + phosphatidyl-(2-O-alpha-D-manno-pyranosyl)-myo-inositol
involved in the early steps of phosphatidylinositol mannoside synthesis, the synthesis of phosphatidylinositol monomannosides and derived higher phosphatidylinositol mannosides in M. smegmatis appears to be dependent on PimA and essential for growth
-
-
?
GDP-mannose + phosphatidylinositol
GDP + phosphatidyl-(2-O-alpha-D-manno-pyranosyl)-myo-inositol
PimA is responsible for the initial mannosylation of phosphatidylinositol
-
-
?
additional information
?
-
-
essential enzyme for mycobacterial growth
-
-
?
additional information
?
-
essential enzyme for mycobacterial growth
-
-
?
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
methyl 6-deoxy-6-dihydroxyphosphinyl-beta-D-galactopyranoside
-
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
phospholipid aggregates
the protein is able to interact with mono-disperse phosphatidylinositol through its active site cleft and also with phospholipid aggregates (micelles or liposomes), possibly through a different region of the protein. The latter interactions stimulate the catalytic activity
-
additional information
-
PimA attaches to the membrane through its N-terminal domain and this association leads to enzyme activation
-
additional information
PimA attaches to the membrane through its N-terminal domain and this association leads to enzyme activation
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
-
enzymatic activity is significantly increased using 1,2-dioctanoyl-sn-glycero-3-phospho-inositol as a substrate
additional information
enzymatic activity is significantly increased using 1,2-dioctanoyl-sn-glycero-3-phospho-inositol as a substrate
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
9.5
calculated, N-terminal half of the protein, residues 1-202
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
PimA attaches to the membrane through its N-terminal domain and this association leads to enzyme activation
sequence contains a putative transmembrane segment, residues 275-298
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
PimA undergoes a conformational reorganization of its N-terminal domain upon phosphatidylinositol membrane interaction.The presence of anionic phospholipids increases the susceptibility of PimA to proteolysis
physiological function
physiological function
both mannosyltransferases PimA and PimB' (MSMEG_4253) recognize phosphatidyl-myo-inositol as a lipid acceptor. PimA specifically catalyzes the transfer of a mannopyranosyl residue to the 2-position of the myo-inositol ring of phosphatidylinositol, whereas PimB' exclusively transfers to the 6-position. PimB' can catalyze the transfer of a mannopyranosyl residue onto the phosphatidylinositol-monomannoside (PIM1) product of PimA, while PimA is unable in vitro to transfer mannopyranosyl onto the PIM1 product of PimB'
physiological function
PimA is involved in the metabolic pathway producing phosphatidylinositol dimannoside PIM2 and phosphatidylinositol hexamannosidePIM6. Crude extracts from Escherichia coli producing recombinant PimA protein synthesize diacylated phosphatidylinositol mono-mannoside from GDP-[14C]Man and bovine phosphatidylinositol. A conditional mutant is unable to grow at the higher temperature at which the rescue plasmid is lost. The synthesis of phosphatidylinositol mono-mannosides and derived higher phosphatidylinositol mannosides appears to be dependent on PimA and essential for growth
physiological function
PimA preferentially binds to negatively charged phosphatidyl-myo-inositol substrate and non-substrate membrane model systems (small unilamellar vesicle) through its N-terminal domain, inducing an important structural reorganization of anionic phospholipids. This interaction is mainly mediated by amphipathic helix alpha2, which undergoes a substantial conformational change and localizes in the vicinity of the negatively charged lipid headgroups and the very first carbon atoms of the acyl chains, at the PimA-phospholipid interface. A flexible region within the N-terminal domain undergoes beta-strand-to-alpha-helix and alpha-helix-to-beta-strand transitions during catalysis and interacts with anionic phospholipids, but the effect is markedly less pronounced to that observed for the amphipathic helix alpha2
physiological function
the stoichiometry of the enzyme-substrate complex strongly depends on phosphatidylinositol concentration. The protein is able to interact with mono-disperse phosphatidylinositol through its active site cleft and also with phospholipid aggregates (micelles or liposomes), possibly through a different region of the protein. The latter interactions stimulate the catalytic activity
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structures of PimA reveal an exceptional flexibility of the protein along the catalytic cycle, including beta-strand-to-alpha-helix and alpha-helix-to-beta-strand transitions. These structural changes modulate catalysis and are promoted by interactions of the protein with anionic phospholipids in the membrane
in complex with GDP and GDP-Man, to 2.4 and 2.6 A resolution, respectively. The structure of PimA in complex with GDP-mannose shows the two-domain organization and the catalytic machinery typical of GT-B glycosyltransferases. Model wherein PimA attaches to the membrane through its N-terminal domain, and this association leads to enzyme activation
in the presence of GDP and myo-inositol, to 2.4 A resolution, orthorhombic space group P212121, one molecule per asymmetric unit
PimA undergoes significant conformational changes upon substrate binding. The binding of the donor GDP-Man triggers an important interdomain rearrangement that stabilizes the enzyme and generates the binding site for the acceptor substrate, phosphatidyl-myo-inositol. The interaction of PimA with the beta-phosphate of GDP-Man is essential for this conformational change. Binding of phosphatidyl-myo-inositol has the opposite effect, inducing the formation of a more relaxed complex with PimA. GDP-Man stabilizes and phosphatidyl-myo-inositol destabilizes PimA by a similar enthalpic amount
crystal structures of PimA in complex with GDP and GDP-Man are determined using multiplewavelength anomalous diffraction methods at 2.4 A and 2.6 A resolution respectively
crystal structures of PimA in complex with GDP and GDP-Man is determined using multiple-wavelength anomalous diffraction methods at 2.4 and 2.6 A of resolution, respectively
sitting-drop vapour-diffusion method. The crystals belong to space group P2(1)2(1)2(1) with unit-cell parameters a = 37.2, b = 72.4, c = 138.2 A and diffract to 2.4 A resolution
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
R77S/K78S/K80S/K81S
mutant is still able to bind GDP with affinities in the submicromolar range but inactive and impaired the ability to bind phospholipid aggregates
W82F W349F
the environment of at least one of the two tryptophan residues (Trp82 and/or Trp349) undergoes structural changes in the presence of membranes
DELTA59-70
mutation of the beta3-beta2 loop: mutant enzyme is still able to bind GDP with affinities in the submicromolar but PimA is completely inactivated and the ability of the protein to bind phospholipid aggregates is drastically impaired
R77S/K78S/K80S/K81S
mutation of the four basic residues on alpha-helix 2: mutant enzyme is still able to bind GDP with affinities in the submicromolar but PimA is completely inactivated and the ability of the protein to bind phospholipid aggregates is drastically impaired
T126W
ability to produce phosphatidylinositol monomannoside (PIM1) is retained, enzymatic activity is similar to wild-type
additional information
additional information
-
a PimA mutant in which the beta3-alpha2 loop is deleted by mutagenesis (PimA5970) is still able to bind GDP with affinities in the submicromolar range but inactive and impaired the abilityto bind phospholipid aggregates
additional information
a PimA mutant in which the beta3-alpha2 loop is deleted by mutagenesis (PimA5970) is still able to bind GDP with affinities in the submicromolar range but inactive and impaired the abilityto bind phospholipid aggregates
additional information
in a polyprotein containing PimA flanked by four copies of the I27 protein, which provides a mechanical fingerprint, PimA exhibits weak mechanical stability albeit displaying beta-sheet topology expected to unfold at much higher forces. PimA unfolds following heterogeneous multiple step mechanical unfolding pathways at low force akin to molten globule states. The open and closed conformations of the GT-B glycosyltransferase are largely present in solution, and in addition, PimA experiences remarkable flexibility that corresponds to the N-terminal Rossmann fold domain
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
GDP-Man stabilizes PimA, phosphatidyl-myo-inositol destabilizes PimA after formation of a more relaxed complex
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
combination of metal ion affinity and anionic exchange and gel filtration chromatography
Talon resin column chromatpgraphy and Superdex 200 gel filtration
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
expression in Escherichia coli BL21(DE3)pLysS cells transformed wth pET-pimA
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
analysis
development of a cell-free assay. Membranes from Mycobacterium smegmatis overexpressing the pimA gene incorporate mannose from GDP-[14C]Man into di- and tri-acylated phosphatidylinositol mono-mannosides
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Guerin, M.E.; Buschiazzo, A.; Kordulakova, J.; Jackson, M.; Alzari, P.M.
Crystallization and preliminary crystallographic analysis of PimA, an essential mannosyltransferase from Mycobacterium smegmatis
Acta Crystallogr. Sect. F
61
518-520
2005
Mycolicibacterium smegmatis, Mycolicibacterium smegmatis (A0QWG6), Mycolicibacterium smegmatis ATCC 700084 (A0QWG6)
Kordulakova, J.; Gilleron, M.; Mikusova, K.; Puzo, G.; Brennan, P.J.; Gicquel, B.; Jackson, M.
Definition of the first mannosylation step in phosphatidylinositol: Mannoside Synthesis. PimA IS essential for growth of mycobacteria
J. Biol. Chem.
277
31335-31344
2002
Mycolicibacterium smegmatis, Mycolicibacterium smegmatis (A0QWG6), Mycolicibacterium smegmatis ATCC 700084 (A0QWG6)
Guerin, M.E.; Kordulakova, J.; Schaeffer, F.; Svetlikova, Z.; Buschiazzo, A.; Giganti, D.; Gicquel, B.; Mikusova, K.; Jackson, M.; Alzari, P.M.
Molecular recognition and interfacial catalysis by the essential phosphatidylinositol mannosyltransferase PimA from mycobacteria
J. Biol. Chem.
282
20705-20714
2007
Mycolicibacterium smegmatis, Mycolicibacterium smegmatis (A0QWG6), Mycolicibacterium smegmatis ATCC 700084 (A0QWG6)
Dinev, Z.; Gannon, C.T.; Egan, C.; Watt, J.A.; McConville, M.J.; Williams, S.J.
Galactose-derived phosphonate analogues as potential inhibitors of phosphatidylinositol biosynthesis in mycobacteria
Org. Biomol. Chem.
5
952-959
2007
Mycolicibacterium smegmatis
Guerin, M.E.; Schaeffer, F.; Chaffotte, A.; Gest, P.; Giganti, D.; Kordulakova, J.; van der Woerd, M.; Jackson, M.; Alzari, P.M.
Substrate-induced conformational changes in the essential peripheral membrane-associated mannosyltransferase PimA from mycobacteria: implications for catalysis
J. Biol. Chem.
284
21613-21625
2009
Mycolicibacterium smegmatis (A0QWG6), Mycolicibacterium smegmatis ATCC 700084 (A0QWG6)
Guerin, M.E.; Kaur, D.; Somashekar, B.S.; Gibbs, S.; Gest, P.; Chatterjee, D.; Brennan, P.J.; Jackson, M.
New insights into the early steps of phosphatidylinositol mannoside biosynthesis in mycobacteria: PimB is an essential enzyme of Mycobacterium smegmatis
J. Biol. Chem.
284
25687-25696
2009
Mycolicibacterium smegmatis (A0QWG6), Mycolicibacterium smegmatis ATCC 700084 (A0QWG6)
Guerin, M.E.; Kordulakova, J.; Alzari, P.M.; Brennan, P.J.; Jackson, M.
Molecular basis of phosphatidyl-myo-inositol mannoside biosynthesis and regulation in mycobacteria
J. Biol. Chem.
285
33577-33583
2010
Mycolicibacterium smegmatis
Giganti, D.; Alegre-Cebollada, J.; Urresti, S.; Albesa-Jove, D.; Rodrigo-Unzueta, A.; Comino, N.; Kachala, M.; Lopez-Fernandez, S.; Svergun, D.I.; Fernandez, J.M.; Guerin, M.E.
Conformational plasticity of the essential membrane-associated mannosyltransferase PimA from mycobacteria
J. Biol. Chem.
288
29797-29808
2013
Mycolicibacterium smegmatis (A0QWG6), Mycolicibacterium smegmatis ATCC 700084 (A0QWG6)
Rodrigo-Unzueta, A.; Martinez, M.A.; Comino, N.; Alzari, P.M.; Chenal, A.; Guerin, M.E.
Molecular basis of membrane association by the phosphatidylinositol mannosyltransferase PimA enzyme from mycobacteria
J. Biol. Chem.
291
13955-13963
2016
Mycolicibacterium smegmatis (A0QWG6), Mycolicibacterium smegmatis ATCC 700084 (A0QWG6)
Giganti, D.; Albesa-Jove, D.; Urresti, S.; Rodrigo-Unzueta, A.; Martinez, M.A.; Comino, N.; Barilone, N.; Bellinzoni, M.; Chenal, A.; Guerin, M.E.; Alzari, P.M.
Secondary structure reshuffling modulates glycosyltransferase function at the membrane
Nat. Chem. Biol.
11
16-18
2015
Mycolicibacterium smegmatis (A0QWG6), Mycolicibacterium smegmatis ATCC 700084 (A0QWG6)
EXTERNAL LINKS (specific for EC-Number 2.4.1.345)
Use of this online version of BRENDA is free under the CC BY 4.0 license. See terms of use for full details.
Release 2023.1 (January 2023)
Legal
Curated at
Member of
