Information on EC 2.3.1.259 - N-terminal methionine Nalpha-acetyltransferase NatF

NatF

acetyl-CoA + an N-terminal-L-methionyl-[transmembrane protein] = an N-terminal-Nalpha-acetyl-L-methionyl-[transmembrane protein] + CoA

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acetyl-CoA:N-terminal-Met-Lys/Ser/Val/Leu/Gln/Ile/Tyr/Thr-[transmembrane protein] Met-Nalpha-acetyltransferase

N-terminal-acetylases (NATs) catalyse the covalent attachment of an acetyl moiety from acetyl-CoA to the free alpha-amino group at the N-terminus of a protein. This irreversible modification neutralizes the positive charge at the N-terminus, makes the N-terminal residue larger and more hydrophobic, and prevents its removal by hydrolysis. NatF is found only in higher eukaryotes, and is absent from yeast. Unlike other Nat systems the enzyme is located in the Golgi apparatus. It faces the cytosolic side of intracellular membranes, and specifically acetylates transmembrane proteins whose N termini face the cytosol. NatF targets N-terminal L-methionine residues attached to Lys, Ser, Val, Leu, Gln, Ile, Tyr and Thr residues.

acetyl-CoA + an N-terminal-L-methionyl-[transmembrane protein]

an N-terminal-Nalpha-acetyl-L-methionyl-[transmembrane protein] + CoA

acetyl-CoA + MAPL

CoA + N-acetyl-MAPL

best substrate

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?

acetyl-CoA + MAPLDLDRWGRPVGRRRRPVRVYP

Nalpha-acetyl-MAPLDLDRWGRPVGRRRRPVRVYP + CoA

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?

acetyl-CoA + MDELFPLRWGRPVGRRRRPVRVYP

Nalpha-acetyl-MDELFPLRWGRPVGRRRRPVRVYP + CoA

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-

?

acetyl-CoA + MKAV

CoA + N-acetyl-MKAV

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-

?

acetyl-CoA + MKGKEEKEGGAR

Nalpha-acetyl-MKGKEEKEGGAR + CoA

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?

acetyl-CoA + MKKSYSGRWGRPVGRRRRPVRVYP

Nalpha-acetyl-MKKSYSGRWGRPVGRRRRPVRVYP + CoA

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?

acetyl-CoA + MKQY

CoA + N-acetyl-MKQY

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?

acetyl-CoA + MLGP

CoA + N-acetyl-MLGP

worst substrate

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?

acetyl-CoA + MLGPEGGRWGRPVGRRRRPVRVYP

Nalpha-acetyl-MLGPEGGRWGRPVGRRRRPVRVYP + CoA

second highest N-terminal acetylation activity

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?

acetyl-CoA + MLGTEGGRWGRPVGRRRRPVRVYP

Nalpha-acetyl-MLGTEGGRWGRPVGRRRRPVRVYP + CoA

highest N-terminal acetylation activity

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?

acetyl-CoA + MVSM

CoA + N-acetyl-MVSM

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?

additional information

?

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5 entries

acetyl-CoA + an N-terminal-L-methionyl-[transmembrane protein]

an N-terminal-Nalpha-acetyl-L-methionyl-[transmembrane protein] + CoA

additional information

?

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the enzyme has a preference for acetylating N-termini of the transmembrane proteins

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acetyl-CoA

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CoA-Ac-MAPLDLD-NH2

very potent inhibitor

CoA-Ac-MKAVQAD-NH2

very potent inhibitor

desulfo-CoA

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0.001

CoA-Ac-MAPLDLD-NH2

Homo sapiens

pH and temperature not specified in the publication

0.00244

CoA-Ac-MKAVQAD-NH2

Homo sapiens

pH and temperature not specified in the publication

0.395

desulfo-CoA

Homo sapiens

pH and temperature not specified in the publication

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-

-

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Naa60 attaches to membranes of the Golgi apparatus. Molecular determinants for the interaction of Naa60 with the membrane by means of computational modeling, in vitro liposome assays including intrinsic tryptophan fluorescence, circular dichroism, and isothermal titration calorimetry (ITC), and mutational analysis combined with localization determination in cellulo. Model of Naa60 membrane association and N-terminal acetylation at the membrane, overview. Peripheral binding to Golgi-like liposomes. Naa60 has a preference for phosphatidylinositol 4-phosphate which may recruit it to the Golgi membrane mainly through Arg212 and Arg215 of Pred-alpha2, and through Lys233 and Arg240 of the C-terminal tail

139

additional information

the C-terminal tail of Naa60 (amino acids 182-242), which constitutes an extra segment, not present in the other catalytic Naas, is defined as the part providing organellar localization. Human eGFP-tagged Naa60 displays an organellar localization recombinantly expressed in Saccharomyces cerevisiae, and this localization pattern is lost and replaced by a cytosolic signal upon truncation of the last 61 amino acids. Naa60 uses a membrane targeting mechanism that is also functional in fungi, although Naa60 is not found in this kingdom. After incubation of the Naa60(189-242) peptide with Golgi-like liposomes composed of 52% phosphatidylcholine, 20% phosphatidylethanolamine, 15% cholesterol, 8% phosphatidylinositol 4-phosphate, and 5% phosphatidylserine, a clear blue shift from 355 to 348 nm is observed, hence indicating an interaction between Naa60(189-242) and these liposomes, residue Trp227 dips into the membrane

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evolution

Nalpha-acetyltransferase 60 (Naa60) belongs to a family of enzymes known as N-terminal acetyltransferases (NATs)

physiological function

additional information

catalytic mechanism of Naa60, structure-function analysis, molecular dynamics simulations, overview

homodimer

x-ray crystallography

hanging drop vapor diffusion method, using 3% (w/v) tacsimate, 0.1 M bis-Tris (pH 6.5), and 16% polyethylene glycol (PEG) 3350

in complex with acetyl-coenzyme A or coenzyme A, hanging drop vapor diffusion method, using 10 mM Tris pH 8.0, 75 mM NaCl, 0.5% (v/v) glycerol, 3% (v/v) Tacsimate pH 4.0, and 7.5% (w/v) polyethylene glycol 3350

D81A

the mutant shows 136.9% catalytic efficiency compared to the wild type enzyme

D81A APPLICATION

the mutant shows about 2fold increased activity compared to the wild type enzyme

D83A

the mutant shows about 2.8fold increased activity compared to the wild type enzyme

E37A

the mutant shows wild type activity

E80A

the mutant shows about 3.6fold increased activity compared to the wild type enzyme

F34A

inactive

H138A

H138F

the mutant is unstable and cannot be purified

H139F

inactive

I167A

the mutant shows an approximate 2.5 to 4fold reduction in kcat and a small increase in KM value, so the catalytic efficiency of this mutant is approximately 3fold of the wild type enzyme

I190A/L191A/Y193A/I194A

site-directed mutagenesis, enzyme deletions suggest membrane interaction by two regions matching the predicted helices (Pred), hydrophobic amino acids in Pred-alpha are mutated. The I190A/L191A/Y193A/I194A mutation, which involves two of the buried amino acids from Pred-alpha1, causes a mixed organellar and cytosolic localization in contrast to the wild-type. This mutation either causes only partially impaired membrane integration of Pred-alpha1 or additional segments are able to maintain some membrane association without Pred-alpha1. Truncation and deletion constructs of eGFP-tagged Naa60 demonstrate regions in the C-terminal tail important for membrane association in cellulo

I209W/W227F

site-directed mutagenesis

I36A

the mutant shows a 6fold reduction in catalytic efficiency compared to the wild type enzyme

I84A

the mutant shows 200.4% catalytic efficiency compared to the wild type enzyme

K79A

the mutant shows about 20% reduced activity compared to the wild type enzyme

K79Q

the mutant shows about 1.8fold increased activity compared to the wild type enzyme

K79R

the mutant shows about 1.5fold increased activity compared to the wild type enzyme

L140A

L197A/L201A/L204A/I209A

site-directed mutagenesis, the mutation results in a mixed localization of the enzyme, with a partial and almost completely extractable organellar fraction, but with a more severe degree of mislocalization compared with mutant I190A/L191A/Y193A/I194A

N143A

the mutant shows about 60% reduced activity compared to the wild type enzyme

P35A

the mutant shows a 6fold reduction in catalytic efficiency compared to the wild type enzyme

P35A/I36A/I167A

inactive

Y164A

the mutant shows 141.7% catalytic efficiency compared to the wild type enzyme

Y164F

the mutant shows 154.9% catalytic efficiency compared to the wild type enzyme

Y165A

inactive

Y165F

the mutant has an approximately 4fold reduction in catalytic efficiency compared to the wild type enzyme

Y193W/W227F

site-directed mutagenesis

Y214W/W227F

site-directed mutagenesis

Y38A

Y97A

inactive

Y97F

additional information

generation of truncated enzyme fragment Naa60(189-242), which peripherally binds Golgi-like liposomes possibly with a horizontal orientation of helices relative to the membrane bilayer

HisTrap column chromatography and Superdex 75 gel filtration

nickel resin column chromatography and Superdex 200 gel filtration

expressed in Escherichia coli BL21 Star cells

expressed in Escherichia coli Rosetta (DE3) pLys cells