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4.2.1.22: cystathionine beta-synthase

This is an abbreviated version!
For detailed information about cystathionine beta-synthase, go to the full flat file.

Word Map on EC 4.2.1.22

Reaction

L-serine
+
L-homocysteine
=
L-cystathionine
 +
H2O

Synonyms

Beta-thionase, CBS, CBS424, CDCP2, CNNM2, Cys4, CysB, cystathionine beta synthase, cystathionine beta-synthase, cystathionine beta-synthase domain-containing protein, cystathionine-beta-synthase, Cysteine synthase, EC 4.2.1.21, hCBS, Hemoprotein H-450, LbrM.17.0230, Methylcysteine synthase, osmoprotectant transporter OpuC, PF1953, PH0267, Serine sulfhydrase, Serine sulfhydrylase, Serine sulphhydrase, TA0289, TM0935, TV1335, yCBS, ytCBS

ECTree

     4 Lyases
         4.2 Carbon-oxygen lyases
             4.2.1 Hydro-lyases
                4.2.1.22 cystathionine beta-synthase

Engineering

Engineering on EC 4.2.1.22 - cystathionine beta-synthase

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A72S
A0A1J9VES8
mutant produces more H2S than wild-type
E220R
A0A1J9VES8
not able to release H2S
A114V
A226T
A331V
-
mutation effects can be suppressed in a yeast assay by the deletion of the regulatory domain of the protein
C15S
-
mutagenesis does not affect catalysis or S-adenosyl-L-methionine activation but significantly reduces aggregation of the purified enzyme in vitro
C165Y
C272A
2fold lower heme content, 2fold lower specific activity, 2fold higher activity in the presence of S-adenosyl-L-methionine
C274S
2fold lower heme content, 2fold lower specific activity, 2fold higher activity in the presence of S-adenosyl-L-methionine
C431S
-
mutagenesis results in a constitutively activated form of CBS that can not be further activated by either S-adenosyl-L-methionine or thermal activation
C52A
-
reduced heme content, pyridoxal phosphate content comparable to wild-type enzyme, low catalytic activity
C52S
-
reduced heme content, pyridoxal phosphate content comparable to wild-type enzyme, low catalytic activity
D198V
pathogenic mutation, no impact on specific activity and response to AdoMet
D234N
the mutation is associated with Venezuelan homocystinuria responsive to vitamin B6. The mutant shows 43% activity compared to the wild type enzyme
D376N
-
mutant is not rescuable by any of the chemical chaperones
DELAT143
-
truncated human CBS lacking 143 amino acids at the C-terminus is purified from a recombinant expression system and is used for vibrational coherence spectroscopy
DELTAC
-
studies are carried out using a truncated protein lacking the C-terminal domain. kcat increases by a factor of 4 and the responsiveness to S-adenosyl-L-methionine is lost. The C-terminal domain is involved in the aggregation of the full-length protein, which exists as a mixture of tetramer and higher oligomers, while the 45 kDa truncated form lacking the C-terminal domain is a dimer
DELTAC143
a truncated human CBS lacking 143 amino acids at the C-terminus is used to study the inactivation of cystathionine beta-synthase by peroxynitrite
E144K
E176K
E201S
mutation leads to permanent activation of enzyme
E302K
G116R
-
mutation in dimer interface of patients with homocystinurea
G148R
G259S
-
active site mutation in patients with homocystinurea
G305R
G307S
H67A
-
mutant is comparable to wild-type, specific activity and Km values for L-Ser, L-homocysteine comparable to wild-type
I152M
-
the mutation is associated with homocystinuria
I278T/T424N
-
mutant enzyme is inactive, although transgenic mouse line that expresses I278T/T424N possess the ability to rescue the neonatal lethality associated with homozygosity for the Cbs- allele
I435T
I437T
mutation results in loss of S-adenosyl-L-methionine-dependent activation but exhibits basal activity that is comparable to that of wild-type enzyme expressed under the same conditions. Purified recombinant I435T shows a two to 3fold higher basal activity compared to wild-type enzyme but is unresponsive to the allosteric activator S-adenosyl-L-methionine
K102N
K523Sfs?18
enzymatic function of the variants is not impaired, increase in the basal enzymatic activity in presence of pyridoxal 5'-phosphate
L539S
-
site-directed mutagenesis, inactive mutant, the mutant shows altered activity compared to the wild-type enzyme
L540Q
enzymatic function of the variants is not impaired, increase in the basal enzymatic activity in presence of pyridoxal 5'-phosphate
N228K
N228S
P422L
P427L
enzymatic function of the variants is not impaired. Mutant lacks activation by S-adenosyl-L-methionine, but binds it at low level and shows an increase in the basal enzymatic activity in presence of pyridoxal 5'-phosphate
P427L/S500L
-
the mutant is almost not activated by S-adenosyl-L-methionine
P78R/K102N
-
KM for L-serine is about 2fold higher than wild-type value. Mutant enzyme is insensitive to allosteric regulation and unresponsive to S-adenosyl-L-methionine
P88S
-
mutation in dimer interface of patients with homocystinurea
Q222X
-
mutagenesis studies reveal that Gln-222 is involved in interactions with substrates
Q526K
-
mutant is not rescuable by any of the chemical chaperones
R125Q
R125W
-
the mutation is associated with homocystinuria
R224A
the mutation decreases CBS activity by approximately 50%
R224H
-
mutation in the connecting loop between the N- and C-terminal domain between beta-strand 7 and alpha-helix 6, patients respond to vitamin B6 treatment
R266G
-
patient mutation , mutant protein shows instability and extensive degradation during thrombin treatment. A GST-R266G fusion protein does not exhibit any detectable activity unlike the GST-tagged wild-type CBS
R266K
R266M
R266X
-
mutagenesis studies reveal that Arg-266 is important to sense structural changes in heme-binding site
R336C
R336H
R369C
R439Q
R491C
-
the mutation is associated with homocystinuria
R51A
the mutation decreases CBS activity by approximately 50%
R58W
-
mutation in the heme binding site of patients with homocystinurea, reduced ability to bind heme
S352N
-
patients with this mutation are not vitamin B6 responsive
S466L
S500L
about 71% of wild-type activity. Mutant lacks activation by S-adenosyl-L-methionine, but binds it at low level and shows an increase in the basal enzymatic activity in presence of pyridoxal 5'-phosphate
T191M
T223X
-
mutagenesis studies reveal that Tyr-223 is involved in interactions with substrates
T257M
-
active site mutation in patients with homocystinurea
T262M
-
expression of human mutant CBS proteins in Saccharomyces cerevisiae reveals that the disease causing mutation severely inhibits enzyme activity and cannot support growth of yeast on cysteine-free media. The osmolyte chemical chaperones glycerol, trimethylamine-N-oxide, dimethylsulfoxide, proline or sorbitol, when added to yeast media, allows growth on cysteine-free media and causes increased enzyme activity from I278T mutant protein. The increase in enzyme activity is associated with stabilization of the tetramer form of the enzyme. This effect is not specific to yeast, as addition of the chaperone glycerol results in increased I278T activity when the enzyme is produced either in Escherichia coli or in a coupled in vitro transcription/translation reaction. No stimulation of specific activity is observed when chaperones are added directly to purified I278T indicating that the presence of chemical chaperones is required during translation
T262R
-
site-directed mutagenesis, inactive mutant
T353M
T434N
-
the mutation is associated with homocystinuria
T87N
the mutation is associated with Venezuelan homocystinuria nonresponsive to vitamin B6. The mutant shows 3.5% activity compared to the wild type enzyme
V180A
V354M
-
patients with this mutation are not vitamin B6 responsive
V371M
-
the mutation is associated with homocystinuria
V449G
enzymatic function of the variants is not impaired, increase in the basal enzymatic activity in presence of pyridoxal 5'-phosphate
W409_G453del
A70S
residue at the substrate binding pocket, important for the H2S-generating activity
E223G
residue at the substrate binding pocket, important for the H2S-generating activity
A70S
-
residue at the substrate binding pocket, important for the H2S-generating activity
-
E223G
-
residue at the substrate binding pocket, important for the H2S-generating activity
-
C272A
-
spectroscopic properties similar to wild-type enzyme
C275S
-
spectroscopic properties similar to wild-type enzyme
T568I
substitution of amino acid residue in the CBS domains of CNNM2, abrogates Mg2+ efflux and binding of ATP
C162S
-
activity can be enhanced by sodium nitroprusside
C367S
-
activity can be enhanced by sodium nitroprusside
C476S
-
activity can be enhanced by sodium nitroprusside
C49S
-
activity can be enhanced by sodium nitroprusside
D249A
-
a series of 8 site-directed mutants is constructed, and their order of impact on the ability of ytCBS to catalyze the beta-replacement reaction is G247S asymptotically equal to K112Q bigger than K112L asymptotically equal to K112R bigger than Y248F bigger D249A asymptotically equal to H138F bigger than E136A
E136A
-
a series of 8 site-directed mutants is constructed, and their order of impact on the ability of ytCBS to catalyze the beta-replacement reaction is G247S asymptotically equal to K112Q bigger than K112L asymptotically equal to K112R bigger than Y248F bigger D249A asymptotically equal to H138F bigger than E136A
G247A
-
undetectable beta-replacement activity
H138F
-
a series of 8 site-directed mutants is constructed, and their order of impact on the ability of ytCBS to catalyze the beta-replacement reaction is G247S asymptotically equal to K112Q bigger than K112L asymptotically equal to K112R bigger than Y248F bigger D249A asymptotically equal to H138F bigger than E136A. Km (L-homocysteine) increased by 8fold
K112L
-
a series of 8 site-directed mutants is constructed, and their order of impact on the ability of ytCBS to catalyze the beta-replacement reaction is G247S asymptotically equal to K112Q bigger than K112L asymptotically equal to K112R bigger than Y248F bigger D249A asymptotically equal to H138F bigger than E136A. Km (L-Ser) increased by 50fold, Km (L-homocysteine) increased by 2fold
K112Q
-
a series of 8 site-directed mutants is constructed, and their order of impact on the ability of ytCBS to catalyze the beta-replacement reaction is G247S asymptotically equal to K112Q bigger than K112L asymptotically equal to K112R bigger than Y248F bigger D249A asymptotically equal to H138F bigger than E136A
K112R
-
a series of 8 site-directed mutants is constructed, and their order of impact on the ability of ytCBS to catalyze the beta-replacement reaction is G247S asymptotically equal to K112Q bigger than K112L asymptotically equal to K112R bigger than Y248F bigger D249A asymptotically equal to H138F bigger than E136A. Km (L-Ser) increased by 90fold, Km (L-homocysteine) increased by 4fold
Q157A
-
no detectable beta-replacement activity, catalyzes a competing beta-elimination reaction, in which L-Ser is hydrolyzed to NH3 and pyruvate
Q157E
-
no detectable beta-replacement activity, catalyzes a competing beta-elimination reaction, in which L-Ser is hydrolyzed to NH3 and pyruvate
Q157H
-
enzyme suffers suicide inhibition via a mechanism in which the released aminoacrylate intermediate covalently attacks the internal aldimine of the enzyme, catalyzes a competing beta-elimination reaction, in which L-Ser is hydrolyzed to NH3 and pyruvate
S289A
-
kcat/Km (L-serine) is reduced by 800fold compared to wild-type. Km (L-homocysteine) equal to wild-type, Km (L-serine) increased compared to wild-type. The reverse-reaction (L-cystathionine hydrolysis) shows a 1400fold reduction of kcat/Km (L-cystathionine) for mutant S289A which is dominated by a 230fold decrease in kcat. Residue S289 is essential in maintaining the properties and orientation of the pyridine ring of the pyridoxal 5'-phosphate cofactor. The reduction in activity of mutant S289A suggests that yeast CBS catalyzes the alpha, beta-elimination of L-Ser via an E1cB mechanism
S289D
-
mutant shows no beta-replacement activity. Fluorescence energy transfer between tryptophan residue(s) of the enzyme and the pyridoxal 5'-phosphate cofactor, observed in the wild-type enzyme and diminished in the S289A mutant, is absent in S289D
S82A
-
catalyzes a competing beta-elimination reaction, in which L-Ser is hydrolyzed to NH3 and pyruvate
T81A
-
catalyzes a competing beta-elimination reaction, in which L-Ser is hydrolyzed to NH3 and pyruvate
T85A
-
catalyzes a competing beta-elimination reaction, in which L-Ser is hydrolyzed to NH3 and pyruvate
Y158F
-
3fold decreased beta-replacement activity, enzyme suffers suicide inhibition via a mechanism in which the released aminoacrylate intermediate covalently attacks the internal aldimine of the enzyme, catalyzes a competing beta-elimination reaction, in which L-Ser is hydrolyzed to NH3 and pyruvate
Y248F
-
a series of 8 site-directed mutants is constructed, and their order of impact on the ability of ytCBS to catalyze the beta-replacement reaction is G247S asymptotically equal to K112Q bigger than K112L asymptotically equal to K112R bigger than Y248F bigger D249A asymptotically equal to H138F bigger than E136A. Km (L-homocysteine) increased by 18fold
additional information