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I312A
mutant retains 30% activity when exposed to oxygen compared to the enzyme assayed under anaerobic conditions
I312A/S363I
mutant retains 75% activity when exposed to oxygen compared to the enzyme assayed under anaerobic conditions
I312S
mutant retains 16% activity when exposed to oxygen compared to the enzyme assayed under anaerobic conditions
I312S/S363I
mutant retains 78% activity when exposed to oxygen compared to the enzyme assayed under anaerobic conditions
I312T
mutant retains 15% activity when exposed to oxygen compared to the enzyme assayed under anaerobic conditions
M295D/I312A
mutant retains 40% activity when exposed to oxygen compared to the enzyme assayed under anaerobic conditions
M295D/I312A/S363I
mutant displays substantially lower levels of activity in comparison to the single as well as double mutant proteins
M295D/I312A/S363V
mutant displays substantially lower levels of activity in comparison to the single as well as double mutant proteins
M295D/I312S
mutant retains 46% activity when exposed to oxygen compared to the enzyme assayed under anaerobic conditions
M295D/I312S/S363I
mutant displays substantially lower levels of activity in comparison to the single as well as double mutant proteins
M295D/I312S/S363V
mutant displays substantially lower levels of activity in comparison to the single as well as double mutant proteins
M295D/I312T
mutant retains 45% activity when exposed to oxygen compared to the enzyme assayed under anaerobic conditions
C172S
-
directed mutagenesis, retains substantial catalytic activity
C256F
-
directed mutagenesis
C256F/K258R/I265V
-
mutant strain grows poorly on minimal medium at 35°C
D473A
-
mutation causes 87% decrease in carboxylation catalytic efficiency and about 15% decrease in CO2/O2 specificity
D94K
-
directed mutagenesis
E43A
-
mutant restores photosynthetic growth to the rbcS knock-out strain, mutant causes decreases in CO2/O2 specificity, Km (CO2) increased compared to wild-type, Km (O2) highly increased compared to wild-type
E92A
-
mutant restores photosynthetic growth to the rbcS knock-out strain, mutant shows a phenotype comparable to wild-type
F81A
-
mutant restores photosynthetic growth to the rbcS knock-out strain, Km (CO2) increased compared to wild-type, Km (O2) highly increased compared to wild-type
G171D
-
directed mutagenesis, recovered by screening acetate-requiring strains
G237S
-
directed mutagenesis, recovered by screening acetate-requiring strains
G344S
-
decrease in CO2/O2 specificity
G54D
-
directed mutagenesis, rbcL missense mutant, recovered by screening acetate-requiring strains
I265V
-
directed mutagenesis
K258R
-
directed mutagenesis
L18A
-
mutant restores photosynthetic growth to the rbcS knock-out strain, mutant affects holoenzyme stability
L290F/V262L
-
directed mutagenesis, increased levels of Rubisco protein observed
L326I
-
directed mutagenesis
L326I/M349L
-
directed mutagenesis
L361I
-
directed mutagenesis, 21% decrease in relative specificity factor
L78A
-
mutant restores photosynthetic growth to the rbcS knock-out strain, Km (CO2) increased compared to wild-type, Km (O2) highly increased compared to wild-type
P19A
-
mutant restores photosynthetic growth to the rbcS knock-out strain, mutant shows a phenotype comparable to wild-type
P79A
-
mutant restores photosynthetic growth to the rbcS knock-out strain, Km (CO2) equal to wild-type, Km (O2) increased compared to wild-type
P89R
-
directed mutagenesis
R217S
-
directed mutagenesis, recovered by screening acetate-requiring strains
S16A
-
mutant restores photosynthetic growth to the rbcS knock-out strain, mutant shows a phenotype comparable to wild-type
T173I
-
directed mutagenesis, recovered by screening acetate-requiring strains
V221C/V235I
-
growth phenotype of mutant strain is indistinguishable from wild-type, kinetic constants are not significantly different from those of wild-type enzyme
V221C/V235I/C256F/K258R/I265V
-
mutant grows significantly better than mutant C256F/K258R/I265V on minimal medium at 35°C
V235I
-
growth phenotype of mutant strain is indistinguishable from wild-type, kinetic constants are not significantly different from those of wild-type enzyme
V331A/G344S
-
altered CO2/O2 specificity
V331A/T342I
-
altered CO2/O2 specificity
W73A
-
mutant restores photosynthetic growth to the rbcS knock-out strain, Km (CO2) increased compared to wild-type, Km (O2) highly increased compared to wild-type
Y32A
-
mutant restores photosynthetic growth to the rbcS knock-out strain, mutant causes decreases in CO2/O2 specificity, Km (CO2) equal to wild-type, Km (O2) decreased compared to wild-type
Y67A
-
the mutant accounts for 10-15fold higher hydrogen production than the wild type enzyme
Y68A
-
the mutant accounts for higher hydrogen production than the wild type enzyme
Y72A
-
the mutant accounts for higher hydrogen production than the wild type enzyme
D473E
-
altered CO2/O2 specificity
-
G344S
-
decrease in CO2/O2 specificity
-
V331A
-
altered CO2/O2 specificity
-
C58S
-
site-directed mutagenesis, elimination of the chemically active site without any loss of activity
D117H
40% reduced CO2/O2 specificity
D117V
40% reduced CO2/O2 specificity
D263A
kcat and Km (substrate: CO2) (substrate: D-ribulose 1,5-bisphosphate) are reduced compared to wildtype
D263E
kcat and Km (substrate: CO2) (substrate: D-ribulose 1,5-bisphosphate) are reduced compared to wildtype, mutant D263E has a significantly higher Km value for (substrate: CO2) compared to mutant D263N (although both mutants have approximately the same kcat value)
D263N
kcat and Km (substrate: CO2) (substrate: D-ribulose 1,5-bisphosphate) are reduced compared to wildtype, mutant D263N has a significantly lower Km value for (substrate: CO2) compared to mutant D263N (although both mutants have approximately the same kcat value)
D263Q
D263Q shows the lowest value for kcat and Km (substrate: CO2) (substrate: D-ribulose 1,5-bisphosphate)
D263S
kcat and Km (substrate: CO2) (substrate: D-ribulose 1,5-bisphosphate) are reduced compared to wildtype
Glu48Carboxymethylcysteine
-
5fold lower specificity factor
H44N
40% reduced CO2/O2 specificity
H44Q
40% reduced CO2/O2 specificity
K122E
-
the mutant is almost inactive (0.8% activity compared to the wild type enzyme)
K122M
-
the mutant is almost inactive (0.2% activity compared to the wild type enzyme)
K122R
-
the mutant with 69% activity compared to the wild type enzyme has a 40% decrease in kcat for carboxylase activity, a 2fold increase in Km for CO2, and a 1.9fold increase in Km for D-ribulose 1,5-bisphosphate
K166C
-
site-directed mutagenesis, able to process RuBP, but the major product is DiMP derived from beta-elimination of phosphate from the enediol
K166C/C58S
-
site-directed mutagenesis, virtually inactive double mutant
K166G
-
site-directed mutagenesis, able to process RuBP, but the major product is DiMP derived from beta-elimination of phosphate from the enediol
K166S
-
site-directed mutagenesis, able to process RuBP, but the major product is DiMP derived from beta-elimination of phosphate from the enediol
M330L
-
increase of Km for carboxylase and oxygenase reaction
A375I
-
mutant shows only insignificant activity and is not able to complement Rhodobacter capsulatus strain SBI/II- (Rubisco knockout strain)
A375S
-
mutant shows only insignificant activity and is not able to complement Rhodobacter capsulatus strain SBI/II- (Rubisco knockout strain)
A375V
-
kcat is highly reduced compared to wild-type, A375V shows 12% activity compared to wild-type, mutant A375V is fully capable of supporting anaerobic phototrophic CO2-dependent growth of the Rhodobacter capsulatus strain SBI/II- (Rubisco knockout strain). Unlike the wild-type Synechococcus enzyme, the A375V mutant is able to support anaerobic photoautotrophic growth at a limiting (1.5%) CO2 concentration. Mutant A375V is able to support aerobic CO2-dependent chemoautotrophic growth and enhance aerobic mixotrophic growth in a complex organic growth media. In contrast to wild-type mutant A375V maintains about 60% of its original activity after a 10 min incubation at 65°C. Thus, the A375V substitution has a pronounced effect on both the structure and function of the enzyme. Km (CO2) value is 23% lower compared to wild-type. Km (O2) value is elevated compared to wild-type. Km (D-ribulose 1,5-bisphosphate) moderately elevated compared to wild-type
D103V
-
mutant fails to support CO2-dependent growth of cultures of Rhodobacter capsulatus since surface residues involved with large subunit interaction influence the Michaelis constant for CO2, kcat is equal to wild-type, mutant D103V shows comparable amounts of Rubisco activity to wild-type after a 10 min incubation at 65°C. Km (CO2) value higher compared to wild-type. Km (O2) and (D-ribulose 1,5-bisphosphate) value equal to wild-type
D103V/A375V
-
kcat is moderately reduced compared to wild-type, mutant D103V/A375V shows 63% activity compared to wild-type,mutant A375V is fully capable of supporting anaerobic phototrophic CO2-dependent growth of the Rhodobacter capsulatus strain SBI/II- (Rubisco knockout strain). The A375V substitution on the same gene as the D103V substitution is an intragenic suppressor that restores photoautotrophic growth when the resulting enzyme is used to complement the Rhodobacter capsulatus strain SBI/II- (Rubisco knockout strain). DOuble mutnat D103V/A375V is to a lesser extent (compared to A375V) able to support aerobic CO2-dependent chemoautotrophic growth and enhance aerobic mixotrophic growth in a complex organic growth media. Mutant D103V/A375V shows comparable amounts of Rubisco activity to wild-type after a 10 min incubation at 65°C. Km (CO2) is moderately elevated compared to wild-type. Km (O2) and (D-ribulose 1,5-bisphosphate) elevated compared to wild-type
F97L
-
mutant is not able to complement Rhodobacter capsulatus strain SBI/II- (Rubisco knockout strain)
F97L/D103V
-
mutant is not able to complement Rhodobacter capsulatus strain SBI/II- (Rubisco knockout strain)
G176D
-
mutant is not able to complement Rhodobacter capsulatus strain SBI/II- (Rubisco knockout strain)
G176D/A375V
-
mutant is not able to complement Rhodobacter capsulatus strain SBI/II- (Rubisco knockout strain). A375V substitution is unable to suppress the negative phenotype of G176D
A340D
-
5% increase in specificity compared to the wild type enzyme
D103E
-
site-directed mutagenesis, mutant enzyme is capable of supporting only slight growth of strain SBI-II- on photoautotrophic plates
D103N
-
site-directed mutagenesis, mutant enzyme is capable of supporting only slight growth of strain SBI-II- on photoautotrophic plates
D103V
-
site-directed mutagenesis, mutant enzyme is capable of supporting only slight growth of strain SBI-II- on photoautotrophic plates
E470P
-
the mutation loses the internal salt bridge and leads to altered surface electrostatics
F15L
-
the mutant shows reduced carboxylation activity compared to the wild type
F342V
-
site-directed mutagenesis, mutant enzyme is shown to support the growth of strain SBI-II- on plates under aerobic chemoautotrophic growth conditions while the wild-type enzyme does not
G122A
-
directed mutagenesis, retains some carboxylase activity
G171A
-
directed mutagenesis, retains some carboxylase activity
G176A
-
2.7fold decrease in turnover number for D-ribulose 1,5-bisphosphate, KM-value for D-ribulose 1,5-bisphosphate is nearly identical to wild-type value
G176D
-
11.7fold decrease in turnover number for D-ribulose 1,5-bisphosphate, 2.2fold increase in KM-value for D-ribulose 1,5-bisphosphate
G176N
-
8.75fold decrease in turnover number for D-ribulose 1,5-bisphosphate, 2fold increase in KM-value for D-ribulose 1,5-bisphosphate
G179A
-
directed mutagenesis, retains some carboxylase activity
G403A
-
directed mutagenesis, retains some carboxylase activity
G405A
-
directed mutagenesis, retains some carboxylase activity
G416A
-
directed mutagenesis, retains some carboxylase activity
G47A
-
directed mutagenesis, retains some carboxylase activity
G47P
-
directed mutagenesis, retains some carboxylase activity
K474T
-
the mutation loses the internal salt bridge and leads to altered surface electrostatics
L138V
-
the mutant shows strongly reduced carboxylation activity compared to the wild type
L27I
-
the mutant shows about wild type carboxylation activity
F15L
-
the mutant shows reduced carboxylation activity compared to the wild type
-
L138V
-
the mutant shows strongly reduced carboxylation activity compared to the wild type
-
L27I
-
the mutant shows about wild type carboxylation activity
-
A340D
-
5% increase in specificity compared to the wild type enzyme
-
D103E
-
site-directed mutagenesis, mutant enzyme is capable of supporting only slight growth of strain SBI-II- on photoautotrophic plates
-
D103N
-
site-directed mutagenesis, mutant enzyme is capable of supporting only slight growth of strain SBI-II- on photoautotrophic plates
-
D103V
-
site-directed mutagenesis, mutant enzyme is capable of supporting only slight growth of strain SBI-II- on photoautotrophic plates
-
E470P
-
the mutation loses the internal salt bridge and leads to altered surface electrostatics
-
F342V
-
site-directed mutagenesis, mutant enzyme is shown to support the growth of strain SBI-II- on plates under aerobic chemoautotrophic growth conditions while the wild-type enzyme does not
-
G176A
-
2.7fold decrease in turnover number for D-ribulose 1,5-bisphosphate, KM-value for D-ribulose 1,5-bisphosphate is nearly identical to wild-type value
-
G176D
-
11.7fold decrease in turnover number for D-ribulose 1,5-bisphosphate, 2.2fold increase in KM-value for D-ribulose 1,5-bisphosphate
-
G176N
-
8.75fold decrease in turnover number for D-ribulose 1,5-bisphosphate, 2fold increase in KM-value for D-ribulose 1,5-bisphosphate
-
K474T
-
the mutation loses the internal salt bridge and leads to altered surface electrostatics
-
C172A
-
point mutagenesis, no decrease in Rubisco content and activities by nitrate deprivation
C172A/192A
-
point mutagenesis, no decrease in Rubisco content and activities by nitrate deprivation
C192A
-
point mutagenesis, stress conditions provoked by nitrate deprivation decreases the Rubisco content and activity
C247A
-
point mutagenesis, stress conditions provoked by nitrate deprivation decreases the Rubisco content and activity
G404A
-
while phosphate-stimulated Rubisco activation diminishes for the mutant, inhibition of catalysis by phosphate remains unchanged. The photosynthesis and growth rate under saturating irradiance and inorganic carbon concentration is not reduced, the mutant shows a marked reductions in kcat compared to the wild type enzyme
H310A
-
turnover number for D-ribulose 1,5-bisphosphate is nearly identical to wild-type value, 1.3fold increase in KM-value for D-ribulose 1,5-bisphosphate
H310D
-
2.9fold decrease in turnover number for D-ribulose 1,5-bisphosphate, 2.8fold increase in KM-value for D-ribulose 1,5-bisphosphate
H327Q
-
5.9fold decrease in turnover number for D-ribulose 1,5-bisphosphate, 3.5fold increase in KM-value for D-ribulose 1,5-bisphosphate, 8.7fold decrease in Ki-value for phosphate
H398A
-
12.7fold decrease in turnover number for D-ribulose 1,5-bisphosphate, 1.9fold increase in KM-value for D-ribulose 1,5-bisphosphate
K305A
-
3.1fold decrease in turnover number for D-ribulose 1,5-bisphosphate, 1.4fold increase in KM-value for D-ribulose 1,5-bisphosphate
K305E
-
2.9fold decrease in turnover number for D-ribulose 1,5-bisphosphate, 1.7fold increase in KM-value for D-ribulose 1,5-bisphosphate
R134A
-
3.1fold decrease in turnover number for D-ribulose 1,5-bisphosphate, 1.5fold increase in KM-value for D-ribulose 1,5-bisphosphate
T65A
-
while phosphate-stimulated Rubisco activation diminishes for the mutant, inhibition of catalysis by phosphate remains unchanged. The photosynthesis and growth rate under saturating irradiance and inorganic carbon concentration is reduced 40-50%, the mutant shows a marked reductions in kcat compared to the wild type enzyme
T65S
-
while phosphate-stimulated Rubisco activation diminishes for the mutant, inhibition of catalysis by phosphate remains unchanged. The photosynthesis and growth rate under saturating irradiance and inorganic carbon concentration is reduced 40-50%, the mutant shows a marked reductions in kcat compared to the wild type enzyme
D69S
site-directed mutagenesis, decameric between 30°C-90°C
E63S
site-directed mutagenesis, decamer at 30°C, gradually dissebles with elevation in temperature
E63S/R66S/D69S
site-directed mutagenesis, is found in a dimeric form even at 30°C
M115F
about 30% decrease in activity
M295D/S363I
significant loss of activity, mutant retains 75% carboxylase activity under oxygen exposed conditions
M295D/S363V
significant loss of activity, mutant retains 75% carboxylase activity under oxygen exposed conditions
M298D
mutant retains 51% carboxylase activity under oxygen exposed conditions
R117A
about 30% decrease in activity
R66S
site-directed mutagenesis, decamer at 30°C, gradually dissebles with elevation in temperature
S363V
mutant retains 71% carboxylase activity under oxygen exposed conditions
S366I
mutant retains 75% carboxylase activity under oxygen exposed conditions
T289D
124% of wild-type activity
V161T
about 10% residual activity
M295D/S363I
-
significant loss of activity, mutant retains 75% carboxylase activity under oxygen exposed conditions
-
M295D/S363V
-
significant loss of activity, mutant retains 75% carboxylase activity under oxygen exposed conditions
-
M298D
-
mutant retains 51% carboxylase activity under oxygen exposed conditions
-
S363V
-
mutant retains 71% carboxylase activity under oxygen exposed conditions
-
S366I
-
mutant retains 75% carboxylase activity under oxygen exposed conditions
-
M295D
-
less sensitive to oxygen compared with the wild-type enzyme, retains 35-40% of activity after exposing to O2
M295D
mutant retains significantly more activity than the wild-type enzyme when incubated with concentrations of oxygen ranging from 10% to 100% in the gas phase. Mutant shows expected competitive inhibition by O2 with respect to CO2
M295D/S363I
-
less sensitive to oxygen compared with the wild-type enzyme, retains 82% of activity after exposing to O2
M295D/S363I
mutant retains significantly more activity than the wild-type enzyme when incubated with concentrations of oxygen ranging from 10% to 100% in the gas phase. Mutant shows expected competitive inhibition by O2 with respect to CO2. The Km value for D-ribulose 1,5-bisphosphate are significantly higher than for the wild-type enzyme
M295D/S363V
-
less sensitive to oxygen compared with the wild-type enzyme, retains 86% of activity after exposing to O2
M295D/S363V
mutant retains significantly more activity than the wild-type enzyme when incubated with concentrations of oxygen ranging from 10% to 100% in the gas phase. Mutant shows expected competitive inhibition by O2 with respect to CO2. The Km value for D-ribulose 1,5-bisphosphate are significantly higher than for the wild-type enzyme
S363I
-
less sensitive to oxygen compared with the wild-type enzyme
S363I
mutant retains significantly more activity than the wild-type enzyme when incubated with concentrations of oxygen ranging from 10% to 100% in the gas phase. Mutant shows expected competitive inhibition by O2 with respect to CO2. The Km value for D-ribulose 1,5-bisphosphate are significantly higher than for the wild-type enzyme
S363V
-
less sensitive to oxygen compared with the wild-type enzyme
S363V
mutant retains significantly more activity than the wild-type enzyme when incubated with concentrations of oxygen ranging from 10% to 100% in the gas phase. Mutant shows expected competitive inhibition by O2 with respect to CO2. The Km value for D-ribulose 1,5-bisphosphate are significantly higher than for the wild-type enzyme
A222T
-
directed mutagenesis
A222T
-
directed mutagenesis, photosynthesis-competent, no substantial deleterious effect on Rubisco function
D473E
-
mutation causes 87% decrease in carboxylation catalytic efficiency and about 15% decrease in CO2/O2 specificity
D473E
-
altered CO2/O2 specificity
L290F
-
directed mutagenesis
L290F
-
directed mutagenesis, recovered by screening acetate-requiring strains
L290F
-
mutation causes 13% decrease in CO2/O2 specificity and reduced thermal stability
L290F/A222T
-
directed mutagenesis, increased levels of Rubisco protein observed
L290F/A222T
-
A222T mutation suppresses the deleterious effects of the L290F mutation to produce a revertant enzyme with improved thermal stability and kinetic properties virtually indistinguishable from that of the wild-type enzyme
M349L
-
directed mutagenesis
M349L
-
directed mutagenesis, 21% decrease in relative specificity factor
T342I
-
directed mutagenesis, 36% decrease in relative specificity factor
T342I
-
altered CO2/O2 specificity
T342I
-
decrease in CO2/O2 specificity
V262L
-
directed mutagenesis
V262L
-
directed mutagenesis, photosynthesis-competent, no substantial deleterious effect on Rubisco function
V331A
-
directed mutagenesis, 42% decrease in relative specificity factor
V331A
-
directed mutagenesis, recovered by screening acetate-requiring strains
V331A
-
altered CO2/O2 specificity
T342I
-
altered CO2/O2 specificity
-
T342I
-
decrease in CO2/O2 specificity
-
L335V
-
directed mutagenesis, 60% decrease in relative specificity factor
L335V
-
directed mutagenesis, 75% decrease in relative specificity factor
V330T
-
negative effect on Rbc activity
V330T
the mutant shows increased activity and reduced thermal stability compared to the wild type enzyme
additional information
-
chimeric small subunits are constructed by replacing the loop of the green alga Chlamydomonas reinhardtii with the sequences of Synechococcus or spinach. When these engineered genes are transformed into a Chlamydomonas mutant that lacks small-subunit genes, photosynthesis-competent colonies are recovered, indicating that loop size is not essential for holoenzyme assembly
additional information
-
the S and L subunits of Rubisco are linked together using a flexible 40-amino acid tether. By replacing the rbcL in tobacco plastids with an artificial gene coding for a S40L fusion peptide, the fusions readily assemble into catalytic (S40L)8 and (S40L)16 oligomers that are devoid of unlinked S subunits. While there is little or no change in CO2/O2 specificity or carboxylation rate of the Rubisco oligomers, their Kms for CO2 and O2 are reduced 10% to 20% and 45%, respectively. In young maturing leaves of the plastome transformants (ANtS40L), the S40L-Rubisco levels are approximately 20% that of wild-type controls despite turnover of the S40L-Rubisco oligomers being only slightly enhanced relative to wild-type
additional information
-
Mutants SP1 and SP2 harbor mutations in the nonconserved residues of loop 6 leading to strongly decreased specific activities. Within alpha-helix 6, SP3 contains a single amino acid substitution from valine to threonine leading to increased specific activity. Mutant SP4 has substitutions in five consecutive amino acid residues leading to increased specific activity. SP5 has a 6-amino-acid substitution in alpha-helix 6 leading to decreased specific activity, while SP6 has 11 substitutions in alpha-helix 6 leading to 31% increased specific activity. In SP7, the entire loop 6/alpha-helix 6 region is replaced with the corresponding region of spinach Rubisco resulting in strongly depressed specific activity.
additional information
comination of mutant T289D and mutant SP8 having a 6-amino-acid substitution in alpha-helix 6 plus mutation V330T gives 151% of wild-type specific activity
additional information
-
comination of mutant T289D and mutant SP8 having a 6-amino-acid substitution in alpha-helix 6 plus mutation V330T gives 151% of wild-type specific activity