Literature summary for 2.7.1.175 extracted from

Fraga, J.; Maranha, A.; Mendes, V.; Pereira, P.J.; Empadinhas, N.; Macedo-Ribeiro, S.
Structure of mycobacterial maltokinase, the missing link in the essential GlgE-pathway (2015), Sci. Rep., 5, 8026.

Search for more literature for 2.7.1.175

Cloned(Commentary)

Cloned (Comment)	Organism
gene mak, DNA and amino acid sequence determination and analysis, phylogenetic analysis, recombinant expression of C-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Mycolicibacterium vanbaalenii
gene mak, phylogenetic analysis, recombinant expression of C-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Mycobacterium tuberculosis

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant enzyme in complex with a non-hydrolysable ATP analogue, mixing of 0.001 ml of protein in 20 mg/ml in 50 mM BTP, pH 7.5, 50 mM NaCl, with 0.0007-0.001 ml of reservoir solution containing 0.1 M MOPS/sodium HEPES, pH 7.5, 0.12 M ethylene glycols (0.03 M each of di-, tri-, tetra-, and penta-ethyleneglycol), 30% PEG 500 MME/PEG 20000, and equilibration against 00.3 ml reservoir solution, 20°C, microseeding, X-ray diffraction structure determination and analysis at 1.15 A resolution, modelling. Crystallzation attempts of the enzyme in complex with maltose are all unsuccessful	Mycolicibacterium vanbaalenii

Crystallization (Comment)

Organism

purified recombinant enzyme in complex with a non-hydrolysable ATP analogue, mixing of 0.001 ml of protein in 20 mg/ml in 50 mM BTP, pH 7.5, 50 mM NaCl, with 0.0007-0.001 ml of reservoir solution containing 0.1 M MOPS/sodium HEPES, pH 7.5, 0.12 M ethylene glycols (0.03 M each of di-, tri-, tetra-, and penta-ethyleneglycol), 30% PEG 500 MME/PEG 20000, and equilibration against 00.3 ml reservoir solution, 20°C, microseeding, X-ray diffraction structure determination and analysis at 1.15 A resolution, modelling. Crystallzation attempts of the enzyme in complex with maltose are all unsuccessful

Mycolicibacterium vanbaalenii

Protein Variants

Protein Variants	Comment	Organism
D334N	inactive mutant	Mycolicibacterium vanbaalenii
D339N	inactive mutant	Mycobacterium tuberculosis
E324R	site-directed mutagenesis, the mutant shows decreased activity compared to the wild-type enzyme	Mycolicibacterium vanbaalenii
E340R	site-directed mutagenesis, the mutant shows decreased activity compared to the wild-type enzyme	Mycobacterium tuberculosis
K413A	site-directed mutagenesis	Mycolicibacterium vanbaalenii
N137A	site-directed mutagenesis, inactive mutant	Mycolicibacterium vanbaalenii
N145A	site-directed mutagenesis, inactive mutant	Mycobacterium tuberculosis
R334R	site-directed mutagenesis, the mutant shows decreased activity compared to the wild-type enzyme	Mycolicibacterium vanbaalenii
R351A	site-directed mutagenesis, the mutant shows decreased activity compared to the wild-type enzyme	Mycobacterium tuberculosis
S136A	site-directed mutagenesis, the mutant shows highly decreased activity compared to the wild-type enzyme	Mycolicibacterium vanbaalenii
S144A	site-directed mutagenesis, the mutant shows highly decreased activity compared to the wild-type enzyme	Mycobacterium tuberculosis
Y416A	site-directed mutagenesis	Mycolicibacterium vanbaalenii
Y416F	site-directed mutagenesis	Mycolicibacterium vanbaalenii
Y420A	site-directed mutagenesis	Mycolicibacterium vanbaalenii
Y420F	site-directed mutagenesis	Mycolicibacterium vanbaalenii

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Mycobacterium tuberculosis
Mg2+	required, magnesium-binding residue is Asp322	Mycolicibacterium vanbaalenii

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
ATP + maltose	Mycolicibacterium vanbaalenii	-	ADP + alpha-maltose-1-phosphate	-	?
ATP + maltose	Mycobacterium tuberculosis	-	ADP + alpha-maltose-1-phosphate	-	?
ATP + maltose	Mycolicibacterium vanbaalenii DSM 7251	-	ADP + alpha-maltose-1-phosphate	-	?
ATP + maltose	Mycobacterium tuberculosis ATCC 25618	-	ADP + alpha-maltose-1-phosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Mycobacterium tuberculosis	O07177	-	-
Mycobacterium tuberculosis ATCC 25618	O07177	-	-
Mycolicibacterium vanbaalenii	A1TH50	-	-
Mycolicibacterium vanbaalenii DSM 7251	A1TH50	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant C-terminally His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity and anion exchange chromatography, followed by gel filtration	Mycolicibacterium vanbaalenii
recombinant C-terminally His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity and anion exchange chromatography, followed by gel filtration	Mycobacterium tuberculosis

Reaction

Reaction	Comment	Organism	Reaction ID
ATP + maltose = ADP + alpha-maltose 1-phosphate	substrate binding structures and catalytic mechanism, overview	Mycolicibacterium vanbaalenii	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + maltose	-	Mycolicibacterium vanbaalenii	ADP + alpha-maltose-1-phosphate	-	?
ATP + maltose	-	Mycobacterium tuberculosis	ADP + alpha-maltose-1-phosphate	-	?
ATP + maltose	-	Mycolicibacterium vanbaalenii DSM 7251	ADP + alpha-maltose-1-phosphate	-	?
ATP + maltose	-	Mycobacterium tuberculosis ATCC 25618	ADP + alpha-maltose-1-phosphate	-	?

Subunits

Subunits	Comment	Organism
More	the N-terminal lobe can be divided into two subdomains: a cap N-terminal subdomain comprising the first 88 amino acid residues and an intermediate subdomain composed of an anti-parallel beta-sheet flanked by two helices. The C-terminal lobe is mostly alpha-helical. While the N-terminal cap subdomain and the C-terminal lobe are predominantly acidic, the intermediate subdomain is enriched in positively charged residues. The N-terminal cap subdomain is composed of three long antiparallel beta-strands forming a curved beta-sheet that encloses the N-terminal alpha-helix and a short two-stranded beta-sheet running perpendicular to the longest beta-sheet axis, on its concave surface. The intermediate subdomain (residues 89-200) contains a central seven-stranded beta-sheet flanked by two alpha-helical segments. A nine-residue linker (residues 201-209) containing a short beta-strand connects the intermediate subdomain and the C-terminal lobe. This last domain is composed of two central 4-helical bundles, a short beta-hairpin and a small two-stranded beta-sheet	Mycolicibacterium vanbaalenii

Synonyms

Synonyms	Comment	Organism
Mak	-	Mycolicibacterium vanbaalenii
Mak	-	Mycobacterium tuberculosis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Mycolicibacterium vanbaalenii
37	-	assay at	Mycobacterium tuberculosis
60	-	-	Mycolicibacterium vanbaalenii

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Mycolicibacterium vanbaalenii
7.5	-	assay at	Mycobacterium tuberculosis

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Mycolicibacterium vanbaalenii
ATP	-	Mycobacterium tuberculosis

General Information

General Information	Comment	Organism
evolution	the enzyme belongs to the family of eukaryotic-like kinases (ELKs) with N-terminal domain topologically resembling the cystatin family of protease inhibitors. Phylogenetic analysis, overview	Mycolicibacterium vanbaalenii
evolution	the enzyme belongs to the family of eukaryotic-like kinases (ELKs) with N-terminal domain topologically resembling the cystatin family of protease inhibitors. Phylogenetic analysis, overview	Mycobacterium tuberculosis
metabolism	the enzyme catalyzes the fourth and last step of the GlcE pathway that channels trehalose to glycogen synthesis and is also likely involved in the biosynthesis of two other crucial polymers: intracellular methylglucose lipopolysaccharides and exposed capsular glucan	Mycolicibacterium vanbaalenii
metabolism	the enzyme catalyzes the fourth and last step of the GlcE pathway that channels trehalose to glycogen synthesis and is also likely involved in the biosynthesis of two other crucial polymers: intracellular methylglucose lipopolysaccharides and exposed capsular glucan	Mycobacterium tuberculosis
additional information	the enzyme shows an eukaryotic-like kinase (ELK) fold, similar to methylthioribose kinases and aminoglycoside phosphotransferases, a typical eukaryotic protein kinase-like fold. Subtle structural rearrangements occur upon nucleotide binding in the cleft between the N- and the C-terminal lobes. The enzyme has a phosphate-binding region in the N-terminal lobe that is proposed to act as an anchoring point tethering maltokinase and trehalose isomerase activities to the site of glycogen biosynthesis, ensuring correct regulation of Mak activity and possibly preventing excessive accumulation of maltose 1-phosphate. The enzyme's unusual N-terminal domain, with the 146AMLKV150 motif, containing the conserved phosphate-binding lysine residue, might regulate its phosphotransfer activity and represents the most likely anchoring point for TreS, the upstream enzyme in the pathway. Putative catalytic base is residue Asp305	Mycolicibacterium vanbaalenii