Open Access

Potential use of sugar binding proteins in reactors for regeneration of CO2 fixation acceptor D-Ribulose-1,5-bisphosphate

  • Sourav Mahato1,
  • Debojyoti De1,
  • Debajyoti Dutta1,
  • Moloy Kundu1,
  • Sumana Bhattacharya2,
  • Marc T Schiavone2 and
  • Sanjoy K Bhattacharya3Email author
Microbial Cell Factories20043:7

DOI: 10.1186/1475-2859-3-7

Received: 08 May 2004

Accepted: 02 June 2004

Published: 02 June 2004

Abstract

Sugar binding proteins and binders of intermediate sugar metabolites derived from microbes are increasingly being used as reagents in new and expanding areas of biotechnology. The fixation of carbon dioxide at emission source has recently emerged as a technology with potentially significant implications for environmental biotechnology. Carbon dioxide is fixed onto a five carbon sugar D-ribulose-1,5-bisphosphate. We present a review of enzymatic and non-enzymatic binding proteins, for 3-phosphoglycerate (3PGA), 3-phosphoglyceraldehyde (3PGAL), dihydroxyacetone phosphate (DHAP), xylulose-5-phosphate (X5P) and ribulose-1,5-bisphosphate (RuBP) which could be potentially used in reactors regenerating RuBP from 3PGA. A series of reactors combined in a linear fashion has been previously shown to convert 3-PGA, (the product of fixed CO2 on RuBP as starting material) into RuBP (Bhattacharya et al., 2004; Bhattacharya, 2001). This was the basis for designing reactors harboring enzyme complexes/mixtures instead of linear combination of single-enzyme reactors for conversion of 3PGA into RuBP. Specific sugars in such enzyme-complex harboring reactors requires removal at key steps and fed to different reactors necessitating reversible sugar binders. In this review we present an account of existing microbial sugar binding proteins and their potential utility in these operations.

Review

Rapid industrialization has led to a dramatically accelerated consumption of fossil fuels with a consequent increase in atmospheric levels of the greenhouse gas carbon dioxide (CO2). This sustained increase of atmospheric CO2 has already initiated a chain of events with negative ecological consequences [13]. Failure to reduce these greenhouse gas emissions will have a catastrophic impact upon both the environment and the economy on a global scale [4, 5]. The reduction has to be brought about by global concerted effort by all countries in order to be effective and meaningful.

At one end of the spectrum – that of generation and utilization of energy resulting in generation of carbon dioxide – hydrocarbons serve as intermediaries for energy storage. Hydrocarbons are not energy by themselves but store energy in their bonds, which is released during combustion. They are thus intermediates for obtaining stored bond energy within them and carbon dioxide is emitted as a consequence of combustion to extract this stored energy. In recent times hydrogen has received renewed attention as the potential replacement for hydrocarbons [610]. However, hydrogen too is an intermediary for obtaining stored bond energy. Recent reports suggest that hydrogen as intermediary may not be entirely free from problems. Also, the problems from use of hydrogen as fuel are yet to be fully realized or foreseen [11, 12]. In all these endeavors a key question, that whether the hydrocarbons will be still retained as intermediaries in energy utilization and the problem of air pollution caused as a result of their combustion can be technologically ameliorated, has not been looked in as much detail as perhaps it should have been. This can possibly be achieved by contained handling of carbon dioxide. The contained handling and fixation of CO2 can be achieved biotechnologically, chemically or by a combination of both.

Sugar binding proteins derived from microbial and other sources have been used for various applications such as diagnostics and affinity purification [13, 14], however they have not been used in environmental biotechnological applications. The possibility of their potential application in environmental biotechnology and review of a few potential candidates is presented here.

The methods in environmental biotechnology that enables efficient capture [15] and fixation of CO2 at emission source/site into concatenated carbon compounds has been pioneered by our group [1619]. The first part in the biocatalytic carbon dioxide fixation is the capture of gaseous CO2. We have pioneered novel reactors employing immobilized carbonic anhydrase for this purpose [15]. Subsequent to capture the carbon dioxide becomes solublized (as carbonic acid or bicarbonate). After adjustment of pH using controllers and pH-stat the solution is fed to immobilized Rubisco reactors [18] where acceptor D-Ribulose-1,5-bisphosphate (RuBP) after CO2 fixation is converted into 3-phosphoglycerate [16, 17]. However, inasmuch as the recycling of acceptor RuBP is central to continuous CO2 fixation, we have invented a novel scheme (Figure 1), which proceeds with no loss of CO2 (unlike cellular biochemical systems) in 11 steps in a series of bioreactors [20]. This scheme is very different from generation of RuBP from D-glucose for start-up process [21] and employing 11 steps in different reactors requiring large volume and weight. The linear combination of reactors with large volume and weight are unsuitable for use with mobile CO2 emitters leaving only the stationary source of emission to be controlled using this technology [17]. To circumvent these problems we have devised a new scheme presented in Figure 2[22]. Based on this scheme, we have designed enzymes as functionally interacting complexes/interactomes or successive conversion in radial flow with layers of uniformly oriented enzymes in concentric circle with axial collection flow system for three enzymes in first reactor for the scheme presented in Figure 2. The four reactors harboring enzymatic complexes/mixtures replace the current 11 reactors. This leads to a faster conversion rate and requires less volume and material weight. However, 4 sugar moieties [3-phosphoglyceraldehyde (3PGAL), Dihydroxyacetone phosphate (DHAP), Xylulose-5-phosphate (X5P) and Ribulose-1, 5-bisphosphate (RuBP)] must be separated at four key steps, as illustrated in Figure 2. In figure 2, using four symbols with solid for bound state and empty for released state, for potential binders: plus for 3PGA, circle for DHAP, cylinder for X5P and box for RuBP, the possible place for utility of these binders have been depicted. In the course of this review, we will consider the availability of enzymatic proteins and non-enzymatic proteins that would be potentially useful as specific binders for these sugar molecules. With a recombinant mutant enzyme we illustrate that such an approach has potential to be used as an in-situ reversible binding matrix for sugar binding and release.
Figure 1

Scheme for generation of D-ribulose-1,5-bisphosphate (RuBP) from 3-phosphoglycerate (3PGA) obtained from fixation of CO2 on RuBP. The continuous regeneration of RuBP in this scheme enables continuous fixation of CO2 at stationary emission sites.

Figure 2

An alternate arrangement of enzymes in the scheme outlined in Fig. 1. This schemes harbors four reactors with indicated enzyme complexes enabling internal channeling, greatly reduces volume and weight for regenerating reactors with faster overall conversion rate to RuBP starting with 3PGA making the system compatible for application in mobile devices in addition to stationary emitters. The reactors may use the sugar binding entities at indicated positions, the hollow and solid symbols represent binding and release phase of the binding-molecules, the plus, circle, cylinder and box are symbols for 3PGA, DHAP, X5P and RuBP binders respectively.

Potential utilizable sugar binding proteins in RuBP regeneration

Three categories of binding proteins can be potentially employed for differential absorption of sugars and for subsequent elution and feeding the reactors downstream in conversion cascade. These are: mutant enzymatic proteins that retain the ability of binding but completely lack any catalytic activity, lectins or proteins of non-immunogenic origin [23] having more than one binding site for the sugar (in nature they cause agglutination of due to sugar binding at multiple sites) and mutant or wild type receptors that binds sugars but are incapable of eliciting further biological activities. The desirable proteins in all these categories are those for which binding affinity is high in a condition close to pH of the emanating solution from the reactor and other conditions for reactor effluent, ability to bind reversibly with respect to some simple but easily manipulable physicochemical parameter (such as temperature, pH, salt concentration), and the ability to be easily attached to a matrix using simple chemistry without loss of binding ability and a long shelf life.

We undertook this review because, although the comprehensive information on a large number of enzymes have been accumulated in BRENDA database [24, 25], but the systematic information on their mutants is lacking and non-enzymatic binders of sugar ligands are not identified / listed in the database.

Proteins that bind 3-phosphoglycerate/3-phosphoglyceraldehye

Both enzymatic and non-enzymatic proteins bind these sugar entities. A number of mutants of many enzymes that bind to either 3-phosphoglycerate or 3-phosphoglyceraldehyde are also known, for example, Phosphoglyceromutase (EC 5.4.2.1), Enolase (EC 4.2.1.11), Mannosyl-3-phosphoglycerate phosphatase (EC 3.1.3.70), Mannosyl-3-phosphoglycerate synthase (EC 2.4.1.217), Phosphoglycerate kinase, (EC 2.7.2.3), Bisphosphoglycerate mutase (EC 5.4.2.4), 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (EC 5.4.2.1), D-3-phosphoglycerate dehydrogenase 2 (EC 1.1.1.95), Cyclic 2,3-diphosphoglycerate-synthetase, Phosphoglycerate dehydrogenase, Transketolase, and Triosephosphate isomerase, BRENDA database shows only three enzymes: Phosphoglycerate dehydrogenase, Mannosyl-3-phosphoglycerate synthase and Phosphoglycerate kinase. A number of mutants of enzymes that binds 3-phosphoglycerate and shows some change in enzymatic activity or kinetic parameters are listed in Table 1. Many of these proteins are reported to retain ligand binding ability with varying degree of loss in catalytic ability (inactive mutants are in bold face), the non-enzymatic protein that also has been reported in literature has been placed towards the bottom part of Table 1. The proteins which retain binding ability but with complete loss in catalytic activity are the ones which warrant further investigation in batch and continuous processes for exploring their suitability as binding proteins in continuous RuBP regenerating reactors (Figure 2). A number of non-enzymatic protein summarized in Table 1 also warrant further exploration. The only binding entity of significance for 3-phosphoglyceraldehyde is 3-phosphoglyceraldehyde dehydrogenase (EC 1.2.1.12) and has not been reviewed.
Table 1

Proteins that bind 3-phosphoglycerate

Source

Mutation

Remarks

References

Enzymatic proteins

Phosphoglycerate mutase 1 (EC 5.4.2.1)

E. coli

Glu327

Lower Vmax

26

S. cerevisiae

Gly13Ser

2-fold increase in activity

27

S. cerevisiae

His181Ala

11-fold increase in the Km

28

S. cerevisiae

C-terminal 7 res. Deletion

Loss of activity, retention of ligand binding

29

B. stearothermophilus

S62A

Loss of activity, retention of ligand binding

30

S. pombe

H163Q

Reduced mutase and phosphatase activities

31

E. coli

R257A

11-fold increase in Vmax

26

E. coli

R307A

700-fold decrease in Vmax

26

Enolase (EC 4.2.1.11)

S. cerrevisiae

S39A

Loss of over 90% activity

32

S. cerrevisiae

H157A, H159A

Loss of over 90% activity

33

S. cerrevisiae

H159A

Loss of over 98% activity

34

Escherichia coli

N341D

Loss of catalytic activity

35

S. cerrevisiae

Gcr1-1 mutation

20-fold reduction in activity

36

Phosphoglycerate kinase, (EC 2.7.2.3)

S. cerrevisiae

H388G

Reduced kcat and Km

37

S. cerrevisiae

R168K

Increase in Km

38

S. cerrevisiae

R168M

Increase in Km

38

S. cerrevisiae

H62D

Increase in Km and Vmax

39

S. cerrevisiae

D372N

reduction in Vmax by 10-folds

40

S. cerrevisiae

R38A

Complete loss of activity

41

S. cerrevisiae

R38Q

Complete loss of activity

41

S. cerrevisiae

R65Q

Increase in Kd, decrease in Km

42

S. cerrevisiae

R65A

Increase in Kd, decrease in Km

42

S. cerrevisiae

R65S

Increase in Kd, decrease in Km

42

S. cerrevisiae

F194W (and F194L)

decrease in Km, Vmax

43

S. cerrevisiae

R203P

Reduction in kcat

44

Bisphosphoglycerate mutase (EC 5.4.2.4)

S. cerevisiae

H181A

Decrease in kcat

28

Transketolase

S. cerevisiae

E418Q, E418A

98–99% reduction in activity

45

S. cerevisiae

E418A

E418 is essential for catalytic activity

45

S. cerevisiae

H103A, H103N and H103F

95–99.9% reduced activity

46

S. cerevisiae

E162A (G)

Impaired catalytic activity and binding

47

S. cerevisiae

D382N(A)

Impaired catalytic activity and binding

47

S. cerevisiae

H481A/S/G

98.5% reduced specific activity

48

S. cerevisiae

N477A

1000-fold decrease in kcat/Km

49

S. cerevisiae

H263A

Reduced activity

50

D-3-phosphoglycerate dehydrogenase 2 (EC 1.1.1.95)

Escherichia coli

L-Serine

Reduced activity

51

Triosephosphate isomerase

Kluyveromyces lactis

Kltpi1 mutant

Loss of activity

52

Plasmodium falciparum

Y74G

Reduced stability

53

Plasmodium falciparum

C13D

7-fold reduction in activity

54

Trypanosoma brucei

W12F

Reduced stability

55

Leishmania mexicana

E65Q

Increased stability

56

K. lactis

DeltaTPI1 mutants

Complete loss of activity

57

Vibrio marinus

A238S mutant

Reduced activity

58

Trypanosoma brucei

C14L

Reduced stability and altered kinetics

59

Saccharomyces cerevisiae

K12R

Vmax reduced by factor of 180

60

Saccharomyces cerevisiae

K12H

No catalytic activity at neutral pH

60

Saccharomyces cerevisiae

E165D

100-fold loss in catalytic activity

61

Salmonella typhimurium

R179L

Reduction in binding affinity

62

Trypanosoma brucei

H47N

Reduced stability

63

Escherechia coli

E165D

100-fold reduction in specific activity

64

Escherechia coli

N78D

Lower kcat

65

Saccharomyces cerevisiae

H95G

400-fold decrease in catalytic activity

66

Non-enzymatic proteins

Phosphoglycerate transporter protein

Salmonella typhimurium

  

67

Salmonella typhimurium

  

68

Bacillus cereus

  

69

Bacillus anthracis

  

70

Salmonella typhi

  

71

Salmonella typhi

  

72

Histone like DNA-binding protein (HU homolog)

Mycobacterium leprae

  

73

Mycobacterium leprae

  

74

Mycobacterium tuberculosis

  

75

Mycobacterium tuberculosis

  

76

40S ribosomal protein SA (P40)

Chlorohydra viridissima

  

77

Strongylocentrotus purpuratus

  

78

Tripneustes gratilla

  

79

Urechis caupo

  

79

Laminin-binding protein

Streptococcus agalactiae

  

80

Streptococcus agalactiae

  

81

Streptococcus pyogenes

  

82

Streptococcus agalactiae

  

83

Streptococcus agalactiae

  

83

Streptococcus agalactiae

  

83

Serine-rich protein (TYE7)

Saccharomyces cerevisiae

  

84

Saccharomyces cerevisiae

85

  

Proteins that bind dihydroxyacetone phosphate

Several enzymes: dihydroxyacetone phosphate acyltransferase, Glycerol-3-phosphate dehydrogenase, Aldolase A, fructose-bisphosphatase, Aldolase B, fructose-bisphosphatase, L-aspartate oxidase, Quinolinate synthetase A, Dihydroxyacetone kinase 1 (Glycerone kinase 1), Glycerol-3-phosphate acyltransferase, NAD(P)H-dependent dihydroxyacetone-phosphate reductase, Dihydroxyacetone phosphate acyltransferase, Alkyl-dihydroxyacetonephosphate synthase, Dihydroxyacetone kinase isoenzyme I, Alpha-glycerophosphate oxidase and Triose phosphate isomerase binds DHAP (Table 2), however, BRENDA shows only four of these proteins, glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), acylglycerone-phosphate reductase (EC 1.1.1.101), glycerone-phosphate O-acyltransferase (EC 2.3.1.42) and alkylglycerone-phosphate synthase (2.5.1.26). The mutants of enzymes with no chemical conversion ability but with high affinity for binding dihydroxyacetone phosphate but very low affinity for other proteins and reversible binding with respect to temperature, salt or pH are desirable properties for the binders.
Table 2

Proteins that bind Dihydroxyacetone phosphate

Source Organism

Mutation

Remarks

References

Enzymatic proteins

Glyceraldehyde-3-phosphate dehydrogenase

S. cerevisiae

ald5 mutant

Higher catalytic activity

86

S. cerevisiae

gpd2 delta mutant

Improved ethanol production

87

Dihydroxyacetone kinase 1 (Glycerone kinase 1)

Hansenula polymorpha

per6-210 mutant

Lacks enzymatic activity

88

Glycerol-3-phosphate acyltransferase

Escherichia coli

G1045A

Reduced specific activity, increased Km

89

Escherichia coli

D311E

Reduced catalytic activity

90

S. cerevisiae

tpa1 mutant

2-fold decrease in activity

91

NAD(P)H-dependent dihydroxyacetone-phosphate reductase

Escherichia coli

Q15R/K, W37R/K

Inactive with NADP+

92

Escherichia coli

Q15K-W37R and Q15R-W37R

30-fold higher Km for NADP+

92

Escherichia coli

gamma-R97Q

10-fold increased Km for NAD

93

Escherichia coli

G252A

Reverse transhydrogenation activity

94

Pseudomonas fluorescens

K295A and K295M

104–106-fold lower turnover

95

M. thermoautotrophicum

R11K and R136K

Decreased Km

96

Alkyl-dihydroxyacetonephosphate synthase

Hansenula polymorpha

ts6 and ts44 mutant

Peroxisomes absent

97

Dihydroxyacetone phosphate acyltransferase

Corynebacterium glutamicum

S187C

Reduced enzymatic activity

98

Triose phosphate isomerase

Kluyveromyces lactis

Kltpi1 mutant

Loss of enzymatic activity

52

Plasmodium falciparum

Y74G

Reduced stability

53, 54

Plasmodium falciparum

C13D

7-fold reduction in the enzymatic activity

53, 54

Trypanosoma brucei

W12F

Reduced stability

55

Leishmania mexicana

E65Q

Increased stability

56

K. lactis

DeltaTPI1 mutants

Complete loss of activity

57

Bacillus stearothermophilus

N12H

Prevent deamidation at high temperature

99

Vibrio marinus

A238S

catalytic activity reduced

58

Trypanosoma brucei

C14L

Reduced stability and altered kinetics

59

Saccharomyces cerevisiae

K12R

Vmax reduced by a factor of 180, Km elevated

60

Saccharomyces cerevisiae

K12H

No catalytic activity at neutral pH

60

Saccharomyces cerevisiae

E165D

1000-fold reduction in catalytic activity

61

Salmonella typhimurium

R179L

Reduction in binding affinity

62

Trypanosoma brucei

H47N

Reduced stability

63

Escherechia coli

E165D

1000-fold reduction in specific activity

64

Escherechia coli

N78D

Lowered Kcat

65

Saccharomyces cerevisiae

H95G

400-fold decrease in catalytic activity

66

Non-enzymatic protein

DHAP transporter

Saccharomyces cerevisiae

  

100

mycoplasma mycoides

  

101

E. coli

  

102

Pseudomonas aeruginosa

  

103

Escherichia coli

  

104

Escherichia coli

  

105

Escherichia coli

  

106

Escherichia coli

107

  

Proteins binding xylulose-5-phosphate

As shown in Table 3 several enzymatic proteins binds to xylulose-5-phosphate. Xylulose-5-phosphate phosphoketolase, Dihydroxyacetone synthase, xylulose kinase, Protein phosphatase 2A B alpha isoform, Xylulose 5-phosphate-activated protein phosphatase, 1-deoxy-D-xylulose 5-phosphate reductoisomerase, 1-deoxy-D-xylulose 5-phosphate synthase 1 and 2 are examples of such enzymes. The non-enzymatic xylulose-5-phosphate binders are shown in the bottom part of Table 3. BRENDA database shows following five proteins, 1-deoxy-D-xylulose-5-phosphate reductoisomerase (EC 1.1.1.267), formaldehyde transketolase (EC 2.2.1.3), 1-deoxy-D-xylulose 5-phosphate synthase (EC 2.2.1.7), Phosphoketolase (EC 4.1.2.9), Ribulose-phosphate 3-epimerase (EC 5.1.3.1).
Table 3

Proteins that bind Xylulose-5-phosphate

Source

Mutation

Remarks

References

Enzymatic proteins

1-deoxy-D-xylulose 5-phosphate reductoisomerase

Escherichia coli

E231K

0.24% wild-type kcat

108

Escherichia coli

H153Q

3.5-fold increase in Km

108

Escherichia coli

H209Q

7.6-fold increase in Km

108

Escherichia coli

H257Q

19-fold increase in Km

108

xylulose kinase

Escherichia coli

XylB- mutant

Lack of growth on xylitol

109

Dihydroxyacetone synthase

Hansenula polymorpha

Pex1–6(ts)

Peroxisome-deficient

110

Hansenula polymorpha

Deltapex14

Lack normal peroxisomes

111

Non-enzymatic proteins

Xylulose-5-phosphate receptor

Mycobacterium tuberculosis

  

112

Xylulose-5-phosphate trasporter

Arabidopsis sp.

  

113

Proteins binding D-Ribulose-1,5-bisphosphate

A number of Ribulose-1,5-bisphosphate and metabolizing enzymes such as Ribulose phosphate kinase and their mutants binds D-ribulose-1,5-bisphosphate. The RuBP binding entities devoid of any enzymatic activities are very valuable in reactors necessitating extraction and separation of RuBP from other sugar compounds (Table 4). Very few non-enzymatic proteins bind RuBP and none of them are microbial sources, and hence have not been incorporated in this review, Rubisco associated protein from soybean is one of them, that show significant RuBP binding [137].
Table 4

Enzymes that bind D-Ribulose-1,5-bisphosphate

Source organism

Mutation

Remarks

References

Rubisco

Chamydomonas reinhardtii

C256F, K258R, L265V

85% decrease in Catalytic efficiency (Vmax/Km)

114

Chamydomonas reinhardtii

G54V

83% decrease in the carboxylation-Vmax

115

Anacystis nidulans

L339F, A340L, S341M

Decrease in Kcat and (Vmax/Km) by 90%and 36.3% respectively

116

Anacystis nidulans

T342I, K343L

Decrease in Kcat and (Vmax/Km) by 90%and 36.3% respectively

116

Anacystis nidulans

T342I

Decrease in Kcat and (Vmax/Km) 40.5%and 40.5% respectively

116

Anacystis nidulans

K343L

Decrease in Kcat and (Vmax/Km) 48.1%and 18.5% respectively

116

Anacystis nidulans

V346Y, D347H, L348T

Inactive

116

Anacystis nidulans

L326I

Decrease in Kcat and (Vmax/Km) 54.4%and 34.2% respectively

116

Anacystis nidulans

S328A

Decrease in Kcat and (Vmax/Km) 5.6%and 41.5% respectively

116

Anacystis nidulans

N123H

16.5% decrease in Kcat

116

Anacystis nidulans

L332M, L332I

>65% decrease in carboxylase but not in oxygenase activity

117

Anacystis nidulans

 

>65% decrease in carboxylase but not in oxygenase activity

117

Anacystis nidulans

L332V

67% decrease in specificity factor (CO2/O2)

117

Anacystis nidulans

L332T

67% decrease in specificity factor (CO2/O2)

117

Anacystis nidulans

L332A

>65% decrease in specificity and carboxylase activity

117

Rhodospirillum rubrum

deleation of F327

99.5% decrease in carboxylase activity

118

Rhodospirillum rubrum

F327L

Increase in Km (RuBP)

118

Rhodospirillum rubrum

F327V

Increase in Km (RuBP)

118

Rhodospirillum rubrum

F327A

Increase in Km (RuBP)

118

Rhodospirillum rubrum

F327G

165-fold increase in Km (RuBP)

118

Rhodospirillum rubrum

N111G

Km(RuBP), kcat are 320 fold increased and 88-fold decreased

119

Rhodospirillum rubrum

N111L

Mutant show a very low carboxylase activity

119

Rhodospirillum rubrum

N111Q

Mutant show a very low carboxylase activity

119

Rhodospirillum rubrum

N111B

Mutant show a very low carboxylase activity

119

Synechococcus sp. PCC6301

I87V

Mutant show a very low carboxylase activity (kcat = 35%)

120

Synechococcus sp. PCC6301

R88K

Mutant show a very low carboxylase activity (kcat = 35%)

120

Synechococcus sp. PCC6301

G91V

Mutant show a very low carboxylase activity (kcat = 35%)

120

Synechococcus sp. PCC6301

F92L

Mutant show a very low carboxylase activity (kcat = 35%)

120

Synechococcus sp. PCC6803

C172A

40–60% decline in Rubisco turnover number

121

Chlamydomonas reinhardtii

N123G

Decrease in specificity factor

122

Chlamydomonas reinhardtii

S379A

Decrease in specificity factor

122

Anacystis nidulans

S376 C

99% and ~99.9% decrease in carboxylase and oxygenase activity

123

Anacystis nidulans

S376T

99% and ~99.9% decrease in carboxylase and oxygenase activity

123

Anacystis nidulans

S376 A

99% and ~16% decrease in carboxylase and oxygenase activity

123

Rhodospirillum rubrum

I164T

6% decrease in carboxylase activity with 40-fold lower Kcat/Km

124

Rhodospirillum rubrum

I164N

1% decrease in carboxylase activity with 900-fold lower Kcat/Km

124

Rhodospirillum rubrum

I164B

0.01–1% decrease in carboxylase activity

124

Rhodospirillum rubrum

H287N

103-fold decrase in carboxylation catalysis

125

Rhodospirillum rubrum

H287Q

105-fold decrase in carboxylation catalysis

125

Rhodospirillum rubrum

M330L

 

126

Rubisco (large subunit)

Chamydomonas reinhardtii

R59A

Decrease in Vmax for carboxylation reaction

127

Chamydomonas reinhardtii

Y67A

Decrease in Vmax for carboxylation reaction

127

Chamydomonas reinhardtii

Y68A

Decrease in Vmax for carboxylation reaction

127

Chamydomonas reinhardtii

D69A

Decrease in Vmax for carboxylation reaction

127

Chamydomonas reinhardtii

R71A

decrease in Vmax (for carboxylation reaction) and thermal stability

127

Chamydomonas reinhardtii

A222T, V262L, L290F

Improved specificity factor and thermal stability

128

Phosphoribulokinase

Rhodobacter sphaeroides

T18A

8-fold decrease in Vmax

129

Rhodobacter sphaeroides

S14A

40-fold decrease in Vmax

129

Rhodobacter sphaeroides

S19A

500-fold and >1500-fold decrease in Vmax and Vmax/Km of RuBP

129

Rhodobacter sphaeroides

K165M, K165C

103-fold decrease in catalytic activity

130

Rhodobacter sphaeroides

R168Q

>300-fold decrease in catalytic efficiency

131

Rhodobacter sphaeroides

R173Q

15-fold decrease in Vmax, 100-fold increase in Km for RuBP

131

Chlamydomonas reinhardtii

R64C

Almost inactive

132

Chlamydomonas reinhardtii

R64A

Decrease in activity

132

Chlamydomonas reinhardtii

R64K

Decrease in activity

132

Synechocystis sp.

S222F

Retains one-tenth catalytic activity

133

Rhodobacter sphaeroides

H45N

40-fold increase in Km for RuBP

134

Rhodobacter sphaeroides

N49Q

200-fold increase in Km for RuBP

134

Rhodobacter sphaeroides

K53M

No effect on catalysis or substrate binding

134

Rhodobacter sphaeroides

D169A

Vmax diminished by 4-orders of magnitude

135

Rhodobacter sphaeroides

D42A

Vmax diminished by 5-orders of magnitude

135

Rhodobacter sphaeroides

D42N

Vmax diminished by 5-orders of magnitude

135

Rhodobacter sphaeroides

R31A

Unlike wild-type, shows hyperbolic kinetics for ATP and NADH

136

Illustrating example

In order to illustrate the utility of non-catalytic enzymatic mutants as specific sugar binders for in-situ separation in reactors, recombinant Saccharomyces cerevisiae 3-phosphoglycerate kinase mutant R38Q [41] was prepared. Mutagenesis was carried out using wild type protein construct in plasmid pET19b as a template. The R38Q mutant was constructed with the Quickchange/Chameleon site-directed mutagenesis kit from stategene using primers as described elsewhere [41]. DNA sequencing of the plasmid identified the mutant. Recombinant wild-type and mutant (R38Q) 3-phosphoglycerate kinase (PGK) were purified to apparent homogeneity as described previously [20] have been shown in Figure 3A. The wild-type and mutant protein was incubated with 10 mM 3-phosphoglycerate barium salt (3PGA) in 50 mM Tris-Cl buffer, pH 7.5 containing 50 mM NaCl for overnight at room temperature. No modification of 3PGA was observed after incubation with R38Q mutant protein (data not shown). The R38Q was coupled with Protein A sepharose beads using dimethylpimelimidate. The recombinant R38Q mutant protein beads (R38Q-PGK) was incubated overnight at room temperature with a mixture of sugars, 3-phosphoglycerate, barium salt (3PGA), ribulose-5-phosphate (R5P), Glucose-6-phosphate (G6P) and Fructose-6-posphate (F1,6-bP) each at a concentration of 10 mM in a volume of 200 μl. After incubation they were washed with 1.5 ml of 180 mM NaCl in 50 mM Tris-Cl buffer, pH 7.5. They were subjected to elution with 1 M NaCl. Lane 1, mixture of sugar prior to incubation with R38Q-PGK and Lane-2 after elution with 1 M NaCl.
Figure 3

The recombinant his-tagged wild-type and R38Q mutant 3-phosphoglycerate kinase was subjected to affinity purification on Ni-NTA column as described previously [20]. A. SDS-PAGE of recombinant wild-type and R38Q mutant S. cerevisiae 3-phosphoglycerate kinase. The proteins (1 and 1.8 μg respectively) was separated in 10% polyacrylamide gel and stained with Coommassie blue R250. B. TLC analysis of sugars prior to and after in-situ separation with R38Q. The recombinant R38Q mutant (R38Q-PGK) was coupled with Protein A sepharose beads and incubated overnight with a mixture of sugars, 3-phosphoglycerate (3PGA), ribulose-5-phosphate (R5P), Glucose-6-phosphate (G6P) and Fructose-6-posphate (F1,6-bP). After washing with 180 mM NaCl, the sugars were eluted with 1 M NaCl. Lane 1, mixture of sugar prior to incubation with R38Q-PGK and Lane-2 after elution with 1 M NaCl.

Conclusion

The enzyme-mutants lacking catalytic activity represent an important group of proteins that could be used for development of sugar-binding proteins reversible with respect to physicochemical parameters such as pH or salt concentration. Nevertheless, the non-enzymatic proteins also represent a suitable repertoire of such potential scaffolds, which could be used for development as sugar-binding proteins to be used in reactors for simultaneous separation of sugars that would be used in subsequent conversion steps. We have developed a RuBP production scheme from 3PGA [16, 17] and also a de novo RuBP production scheme from D-glucose [21] for continuous CO2 fixation and for start-up of the fixation respectively employing series of reactors. Both systems for production of RuBP will benefit from specific sugar binders but besides their use in environmental biotechnology, they will find application in diagnostics, separation technologies and also as research reagents.

Declarations

Acknowledgements

We thank Dr. Paramita Ray for help with literature search and Dr. Surabhi Choudhuri for her comments on the manuscript.

Authors’ Affiliations

(1)
Department of Biotechnology, Haldia Institute of Technology
(2)
Environmental Biotechnology Division, ABRD Company LLC
(3)
Department of Ophthalmic Research, Cleveland Clinic Foundation

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