6.1.1.11: serine-tRNA ligase

the mutant shows increased activity compared to the wild type enzyme

C46S

the mutant shows increased activity compared to the wild type enzyme

DELTAG254-N261

the mutant shows 72% activity compared to the wild type enzyme

728813

DELTAG75-N97

the mutant shows 13% activity compared to the wild type enzyme

728813

DELTAG75-N97/DELTAG254-N261

the mutant shows 7% activity compared to the wild type enzyme

728813

E156T/R157S

the mutant retains about 10% of wild type activity

E447K

the mutant shows increased activity compared to the wild type enzyme

G136V

the mutant retains about 15% of wild type activity

P30G31Y

the mutation of the P30G31 dipeptide to a single tyrosine almost eliminates serylation activity

Methanococcus maripaludis

C315A

site-directed mutagenesis

715080

E273A

E273A replacement in helix 9 located in the C-terminal domain

E273A/D277A/K280A

triple replacement in helix 9

E338A

Km (mM)(tRNASer): 0.0021, kcat (1/sec) (tRNASer): 0.059

F397P/A399G

the mutant shows deleterious effects in both kcat and KM values compared to the wild type enzyme

G340V/G341A

Km (mM)(tRNASer): 0.0067, kcat (1/sec) (tRNASer): 0.000000011

G402A/G405A

the double substitution displays an order of magnitude higher KM but only 2.5fold lower kcat

H250A

the mutant is highly efficient in serine activation: kcat is reduced by only 3fold, while KM is reduced relative to the wild type parameters

H250A/Q400A

the mutant is highly efficient in serine activation: kcat is reduced by only 3fold, while KM is reduced relative to the wild type parameters

K141A

mutated enzyme, serylation rate lowered

K164A

K164A replacement in the linker peptide

K79A

mutated enzyme, significant drop in the serylation rate

K87A

mutated enzyme, retains the serylation ability comparable with the wild-type enzyme

K88A

mutated enzyme, retains the serylation ability comparable with the wild-type enzyme

K90A

mutated enzyme, retains the serylation ability comparable with the wild-type enzyme

mC306A

active site mutant

mC306A/C461A

active site mutant

mE338A

active site mutant

mG340V/G341A

active site mutant

mMbSerRS-CTD

START codon introduced at position 167, which generates the C-terminal domain

mMbSerRS-CTDDELTAHTH

START codon introduced at position 167 of mMbSerRS-DELTAHTH, generating the C-terminal domain without the helix-turn-helix motif

mMbSerRS-DELTAHTH

deleted helix-turn-helix motif

mMbSerRS-NTD

STOP codon introduced at position 164, which generates the N-terminal domain

mR267A

active site mutant

mR347A

active site mutant

mW396A

active site mutant

N142A

mutated enzyme, serylation rate lowered

N435A

Km (mM) (L-Ser): 0.07028, kcat (1/sec) (L-Ser): 0.172, Km (mM)(tRNASer): 0.283, kcat (1/sec) (tRNASer): 0.435

N435A/S437A

Km (mM) (L-Ser): 0.689, kcat (1/sec) (L-Ser): 0.576, Km (mM)(tRNASer): 0.509, kcat (1/sec) (tRNASer): 0.361

N435A/S437A/W396A

Km (mM) (L-Ser): 0.2879, kcat (1/sec) (L-Ser): 1.32, Km (mM)(tRNASer): 1.191, kcat (1/sec) (tRNASer): 0.175

P395A

the mutant shows 6.5fold reduced enzyme efficiency compared to the wild type

Q400A

the mutation produces only small kinetic effects: KM is elevated 2fold while kcat is reduced 2fold

R143A

mutated enzyme, serylation rate lowered

R147A

R147A replacement in helix 4 located in the N-terminal domain

R267A

Km (mM)(tRNASer): 0.0034, kcat (1/sec) (tRNASer): 0.032

R347A

Km (mM)(tRNASer): 0.0063, kcat (1/sec) (tRNASer): 0.0026

R38A

mutated enzyme, serylation rate lowered

R76A

mutated enzyme, serylation ability completely lost

R78A

mutated enzyme, retains the serylation ability comparable with the wild-type enzyme

R94A

mutated enzyme, significant drop in the serylation rate

S437A

Km (mM) (L-Ser): 0.573, kcat (1/sec) (L-Ser): 0.729, Km (mM)(tRNASer): 0.412, kcat (1/sec) (tRNASer): 0.396

W396A

Km (mM)(L-Ser): 0.2645, kcat (1/sec) (L-Ser): 0.287, Km (mM)(tRNASer): 0.760, kcat (1/sec) (tRNASer): 0.239

Y89A

mutated enzyme, retains the serylation ability comparable with the wild-type enzyme

A289V

mutations in the active site mutant SerRS11, mutant is less sensitive to inhibition by serine hydroxamate and 5'-O-[N-(L-seryl)-sulfamoyl]adenosine

D288Y

mutations in the active site mutant SerRS11, mutant is less sensitive to inhibition by serine hydroxamate and 5'-O-[N-(L-seryl)-sulfamoyl]adenosine

E281D/G291A

reduced activity compared to the wild-type enzyme

651138

K287R

mutations in the active site mutant SerRS12, mutant is less sensitive to inhibition by serine hydroxamate and 5'-O-[N-(L-seryl)-sulfamoyl]adenosine

K287T

mutations in the active site mutant SerRS11, mutant is less sensitive to inhibition by serine hydroxamate and 5'-O-[N-(L-seryl)-sulfamoyl]adenosine

ScSerRSDELTAC13

mutant containing a deletion of 13 C-terminal residues, mutant is readily complement a ScSerRS null allele, Km and Kcat values comparable to wild-type, T50: 44.5°C, interaction with Pex21p is lost

ScSerRSDELTAC203

mutant containing a deletion of 20 C-terminal residues, mutant does not complement a ScSerRS null allele, T50: 44.5°C

H270G

SerRS2 mutant

DELTA2-22

in transformed protoplasts, transient expression of deletion mutant reveals GFP fluorescence throughout the cytosol, implying that the very N-terminal region is necessary for the correct targeting of SerZMo into mitochondria and chloroplasts

694659

DELTA2-73

transient expression of the SerZMo fusion protein with fully truncated N-terminal extension DELTA2-73 SerZMo-GFP, give diffuse GFP fluorescence throughout the cytosol and shos no association with mitochondria or chloroplasts

694659

DELTA81-511

mutant construct containing only the first 80 amino acids is expressed in chloroblasts and in mitochondria as shown by GFP-fusion protein

694659

ZmcSerRSDELTAC12

mutant containing a deletion of 12 C-terminal residues, mutant readily complements a yeast SerRS null allele, Km and Kcat values comparable to wild-type

ZmcSerRSDELTAC18

mutant containing a deletion of 18 C-terminal residues, mutant readily complements a yeast SerRS null allele, Km and Kcat values comparable to wild-type, T50: 31.5°C, interaction with Pex21p is lost

ZmcSerRSDELTAC26

mutant containing a deletion of 26 C-terminal residues, mutant does not complement a yeast SerRS null allele, enzymatic activity of mutant protein is comparable to negative control. Expression of mutant protein is probably severly hindered due to intrinsic instability of the truncated protein