Hyperthermophilic Carbamate Kinase Stability and Anabolic In Vitro Activity at Alkaline pH

34 Pages·2017·3.83 MB·English

AEM Accepted Manuscript Posted Online 17 November 2017
Appl. Environ. Microbiol. doi:10.1128/AEM.02250-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
1 Hyperthermophilic Carbamate Kinase Stability and Anabolic In Vitro Activity at
2 Alkaline pH
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4 James E. Hennessy,a Melissa J. Latter,a Somayeh Fazelinejad,a Amy Philbrook,a Daniel M.
5 Bartkus,a Hye-Kyung Kim,a Hideki Onagi,a John G. Oakeshott,b Colin Scott,b Apostolos
6 Alissandratos,a Christopher J. Eastona D
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8 Research School of Chemistry, Australian National University, Canberra, Australiaa; CSIRO ad
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9 Land and Water, Black Mountain Laboratories, Canberra, Australiab f
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11 Running Head: In Vitro Anabolic Activity of Carbamate Kinases :
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12 m
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13 Address correspondence to Christopher J. Easton, [email protected] or Apostolos s
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14 Alissandratos, [email protected]. r
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15 Abstract. Carbamate kinases catalyze the conversion of carbamate to carbamoyl
16 phosphate, which is readily transformed into other compounds. Carbamate forms
17 spontaneously from ammonia and carbon dioxide in aqueous solutions, so the kinases have
18 potential for sequestrative utilization of the latter compounds. Here, we compare seven
19 carbamate kinases from mesophilic, thermophilic and hyperthermophilic sources. In addition
20 to the known enzymes from Enterococcus faecalis and Pyrococcus furiosus, the previously D
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21 unreported enzymes from the hyperthermophiles Thermococcus sibiricus and Thermococcus n
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22 barophilus, the thermophiles Fervidobacterium nodosum and Thermosipho melanesiensis, d
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23 and the mesophile Clostridium tetani, were all expressed recombinantly, each in high yield. f
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24 Only the clostridial enzyme did not show catalysis. In direct assays of carbamate kinase
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25 activity, the three hyperthermophilic enzymes display higher specific activities at elevated :
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26 temperatures, greater stability, and remarkable substrate turnover at alkaline pH (9.9-11.4). m
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27 Thermococcus barophilus and Thermococcus sibiricus carbamate kinase were found to be the s
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28 most active when the enzymes were tested at 80 °C, and maintain activity over broad r
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29 temperature and pH ranges. These robust thermococcal enzymes therefore represent ideal n

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30 candidates for biotechnological applications involving aqueous ammonia solutions, since ar
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31 non-buffered 0.0001-1.0 M solutions have pH values of approximately 9.8-11.8. As proof-of- 2
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32 concept, here we also show that carbamoyl phosphate produced by the Thermococcus 0
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33 barophilus kinase is efficiently converted in situ to carbamoyl aspartate, by aspartate b
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34 transcarbamoylase from the same source organism. Using acetyl phosphate to simultaneously u
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35 recycle the kinase cofactor ATP, carbamoyl aspartate is produced directly from solutions of t
36 ammonia, carbon dioxide and aspartate, in high yield at pH 9.9.
37 Importance. Much of the nitrogen in animal wastes and used in fertilizers is commonly
38 lost as ammonia in water run-off, from which it must be removed to prevent downstream
39 pollution and evolution of nitrogenous greenhouse gases. Since carbamate kinases transform
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40 ammonia and carbon dioxide to carbamoyl phosphate via carbamate, and carbamoyl
41 phosphate may be converted into other valuable compounds, the kinases provide a route for
42 useful sequestration of ammonia, as well as carbon dioxide, another greenhouse gas. At the
43 same time, recycling the ammonia in chemical synthesis reduces the need for its energy-
44 intensive production. However, robust catalysts are required for such biotransformations.
45 Here we show that carbamate kinases from hyperthermophilic archaea display remarkable D
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46 stability and high catalytic activity across broad ranges of pH and temperature, making them n
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47 promising candidates for biotechnological applications. We also show that carbamoyl d
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48 phosphate produced by the kinases may be efficiently used to produce carbamoyl aspartate. f
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49 INTRODUCTION p
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50 Carbamoyl phosphate (CP) is an important metabolic intermediate. It is produced in e
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51 anabolic pathways through the action of carbamoyl phosphate synthetases, consuming two s
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52 equivalents of ATP, and normally degraded in catabolic pathways through the action of r
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53 carbamate kinases (CKs), for the generation of one equivalent of ATP (1). The kinase activity on

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54 is reversible, however, and CKs are able to catalyze CP formation when supplied with ATP a
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55 and carbamate. Carbamate forms spontaneously in aqueous solutions of ammonia and carbon 2
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56 dioxide (Figure 1) (2). 2
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57 CK was first isolated from Enterococcus faecalis where it catalyzes ATP production as
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58 part of the arginine dihydrolase fermentation pathway (3). The kinase gene has since been g
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59 identified in various other species that derive energy from arginine degradation (4-6). CKs t
60 have also been purified from pathogens of medical interest such as Mycoplasma penetrans
61 (7), Giardia lamblia (8) and Trichomonas vaginalis (9). In the hyperthermophilic archaeon
62 Pyrococcus furiosus, an enzyme originally described as a CK-like carbamoyl phosphate
63 synthetase was subsequently characterized to be a CK (1, 10, 11). It is proposed to naturally
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64 catalyze anabolic CP formation (12, 13). This is associated with the absence of the arginine
65 dihydrolase pathway in Pyrococcus furiosus and the relatively high concentrations of
66 ammonia and carbon dioxide, and therefore carbamate, in hydrothermal vents where this
67 organism thrives (11, 12).
68 We were interested in the potential in vitro use of CKs in the anabolic direction, for the
69 conversion of ammonia and carbon dioxide into CP. CP is readily transformed, biologically D
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70 and chemically, into other useful compounds (14-19). Therefore, this application of CKs is of n
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71 interest as a method for the biocatalytic capture and re-use of waste ammonia. It would d
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72 involve relatively high concentrations of ammonia, and therefore alkaline conditions. f
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73 Consequently, we were particularly focused on pH stability and activity, as well as overall
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74 robustness, of CKs from different source organisms. :
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75 Herein, along with the earlier reported enzymes from Pyrococcus furiosus (CK-Pf) and the m
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76 mesophile Enterococcus faecalis (CK-Ef), previously unreported CKs from Thermococcus s
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77 sibiricus (CK-Ts), Thermococcus barophilus (CK-Tb), Fervidobacterium nodosum (CK-Fn), r
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78 Thermosipho melanesiensis (CK-Tm) and Clostridium tetani (CK-Ct) were each produced. n

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79 All enzymes were expressed recombinantly using an optimized protocol, which led to 1,000- ar
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80 fold higher protein yields compared to CK-Pf isolated from Pyrococcus furiosus (10). The 2
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81 activity of CK-Pf was previously measured using a coupled enzyme assay, which indicated a 0
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82 pH optimum of around 8.0 (10). We developed a direct assay of substrate turnover by CKs, b
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83 applicable for use with any ATP-dependent enzyme. This instead shows that CK-Pf displays u
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84 remarkable stability and activity at alkaline pH (> 11), with maximum catalysis at above pH t
85 9.4. The difference between our results and those reported is attributable to complications
86 associated with the earlier protocol. Both CK-Ts and CK-Tb showed stability and activity at
87 alkaline pH similar to that displayed by CK-Pf. This activity under alkaline conditions was
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88 not displayed by the mesophilic and thermophilic CKs tested here and is atypical of
89 intracellular enzymes (20).
90 The utility of the production of CP by these CKs depends on having the ability to use the
91 CP to make other compounds. Although CP decomposes in alkaline aqueous solutions (21),
92 for example with a half-life of 10 mins at pH 9.9 and 37 °C, as a metabolic intermediate it is
93 processed in vivo by transcarbamoylases (22), that limit CP breakdown through metabolic D
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94 channeling (23) and conformational restriction (24). Here, we establish the viability of using n
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95 a similar approach in vitro, through in situ conversion of CP produced by CK-Tb into d
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96 carbamoyl aspartate, by Thermococcus barophilus aspartate transcarbamoylase (ATC-Tb). f
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97 Using E. coli lysate and acetyl phosphate (AP) to recycle the kinase cofactor ATP (25) during
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98 this process, a solution of ammonia, carbon dioxide and aspartate, at pH 9.9, efficiently :
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99 produces a high yield of carbamoyl aspartate. m
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100 RESULTS AND DISCUSSION r
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101 Selection and recombinant expression of carbamate kinases. To investigate the on

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102 biocatalyst space, in addition to the already reported hyperthermophilic CK-Pf and a
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103 mesophilic CK-Fn, CKs from five alternative source organisms were selected. Organisms of 2
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104 the genus Thermococcus are hyperthermophilic archaea that form separate clusters to those of 2
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105 the genus Pyrococcus in the order of Thermococcales (26). For Thermococcus sibiricus (27)
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106 and Thermococcus barophilus (26), genomic analysis shows a lack of arginine dihydrolase g
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107 and the presence of pyrimidine and arginine biosynthetic enzymes. This suggests that CK-Ts t
108 and CK-Tb may play a similar anabolic physiological role to CK-Pf. Accordingly, CK-Ts and
109 CK-Tb were selected, to compare their activity with that of CK-Pf, with which they each
110 share 77% amino acid sequence similarity. CK-Fn and CK-Tm from the thermophilic bacteria
111 Fervidobacterium nodosum and Thermosipho melanesiensis, each with 48% sequence
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112 similarity to CK-Pf, were also targeted to more generally examine the behavior of CKs from
113 thermophiles. Finally, CK-Ct from Clostridium tetani was chosen to include another CK from
114 a non-thermophilic source. CK-Pf shares 42% and 50% sequence similarity with CK-Ef and
115 CK-Ct, respectively. An alignment of the amino acid sequences of the seven carbamate
116 kinases investigated is presented in Figure 2.
117 The reported isolation of CK-Pf gave a low protein yield of only 15 μg per g of cell pellet. D
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118 A higher yield has been obtained through recombinant expression in E. coli, but only by n
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119 following an elaborate protocol with multiple protein purification steps (11). This also d
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120 required the simultaneous over-expression of archaeal tRNA in the bacterial host due to f
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121 direct use of the archaeal DNA sequence. For the present work, the CK genes from
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122 Pyrococcus furiosus and the other selected source organisms were instead codon-optimized :
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123 for expression in E. coli. Each was encoded with an N-(His)6-tag to allow easy purification m
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124 through Ni-affinity chromatography. That resulted in production of 16 mg of CK-Pf per g of s
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125 cell pellet. This protein yield represents a 1,000-fold improvement compared to isolation of r
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126 the enzyme from the source organism (10), and a 3-fold improvement with a much-simplified n

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127 protocol compared to the earlier recombinant co-expression (11). CK-Ts, CK-Tb, CK-Fn, ar
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128 CK-Tm, CK-Ef and CK-Ct were each expressed in similar yield to CK-Pf. SDS-PAGE 2
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129 analysis following Ni-affinity chromatography confirmed the presence of purified, 0
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130 overexpressed protein in all cases (Figure 3). Differences between the mobilities of the CKs b
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131 on the gel may be attributed to a range of factors, such as variations between the protein u
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132 aliphatic indices and hydropathy (GRAVY) values, which ranged from 88.69 to 97.80 t
133 and -0.113 to -0.350, respectively, when calculated using ProtParam (ExPASy server).
134 Determination of carbamate kinase activity. The previously reported assay of CK-Pf
135 activity used a coupled enzyme method, with indirect measurement of the ADP formed to
136 assess CP production (10). It relied firstly, on the reaction of ADP with phosphoenolpyruvate
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137 to produce ATP and pyruvate, catalyzed by pyruvate kinase, and secondly, the lactate
138 dehydrogenase-catalyzed, NADH-dependent reduction of the pyruvate to lactate. The
139 decrease in NADH concentration was then monitored through measuring the UV absorbance
140 at 340 nm. In our study, an HPLC-UV method was instead developed to directly analyze
141 ADP formation and CK activity. It is based on the separation and detection of ATP, ADP and
142 AMP, which all absorb at 259 nm. Therefore, in principle, it may be used to monitor any D
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143 ATP-, ADP- or AMP-dependent enzyme, and is suitable for automation. The assay was used n
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144 to study the behavior of the recombinantly expressed CKs under a range of conditions, with d
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145 variations between our observations and those reported earlier for CK-Pf being a direct f
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146 consequence of us being able to avoid the confounding effects of coupled enzymes.
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147 Initially, CK catalysis was studied in aqueous solutions prepared using 0.2 M NaHCO3, :/
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148 0.2 M NH3, 10 mM ATP and 10 mM MgCl2, adjusted to between pH 7.9-11.4 with 6 M m
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149 NaOH or HCl. Carbamate then forms spontaneously according to the equations shown in s
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150 Figure 1. Studies were not carried out at lower or higher pH, as this is beyond the reliable r
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151 buffering capacity of ammonia and bicarbonate. Within this range, the pH of solutions varies n

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152 by less than 0.1 during the course of the assays. Representative results from assays under ar
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153 these conditions at 40 °C are illustrated in Figure 4. Of the seven overexpressed CKs, only 2
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154 CK-Ct did not show activity. Thus, like those of CK-Pf and CK-Ef, the genes for CK-Ts, CK- 0
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155 Tb, CK-Tm and CK-Fn were established to encode active kinases. Surprisingly, CK-Pf was b
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156 found to be active over the entire pH range studied, with optimum activity at pH 9.9 and u
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157 above (Figure 4a). CK-Ts and CK-Tb showed higher specific activities, but the same increase t
158 in activity with increase in pH (Figures 4b,c). This activity at high pH is not shared by the
159 non-hyperthermophilic CKs. CK-Fn exhibits a pH optimum of 9.9 but a sharp decrease in
160 activity at higher pH, with little activity at pH 10.9 or above (Figure 4d). Initial screening
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161 showed that the pH activity profiles of CK-Tm and CK-Ef (data not shown) are similar to
162 those of CK-Fn, so those kinases were not studied in detail.
163 The stability and activity of the three hyperthermophilic proteins at high pH, with its
164 potential relevance to processing ammonia from alkaline waste streams, instead prompted us
165 to further examine the pH activity-dependence of these CKs. Accordingly assays were also
166 carried out with solutions prepared using 0.05 M NH3 and NaHCO3, as well as 0.4 M of each D
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167 reagent, in addition to the 0.2 M quantities discussed above. The results of these analyses at n
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168 40 °C, are presented in Figure 5. The enzyme kinetics are complex, with the binding and d
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169 reaction of both carbamate and ATP all probably pH-dependent. Compounding this, the f
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170 concentration of carbamate available for catalysis is dependent on the quantities of ammonia
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171 and bicarbonate used to prepare the assay solutions, and pH-dependent equilibria between the :
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172 ammonia and bicarbonate, as well as carbon dioxide and ammonium hydroxide (Figure 1). m
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173 Given this complexity, only general conclusions should be drawn from the results. Even so, s
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174 for each of the hyperthermophilic CKs, the shapes of the pH profiles at the different reagent r
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175 concentrations (Figure 5) are broadly similar. At each concentration, the CKs all show high n

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176 activity at pH 9.4 and above. ar
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177 The most obvious general effect of increasing and decreasing the concentrations of 2
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178 ammonia and bicarbonate is a matching rise and fall in enzyme activity. This is attributable to 0
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179 corresponding changes in the concentration of carbamate, showing that the kinases are not b
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180 saturated with this substrate. Under these circumstances, it is even more remarkable that the u
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181 hyperthermophilic kinases retain their activity at the highest pH values, where there is less t
182 carbamate. We used 13C NMR spectroscopy to measure carbamate concentration as a
183 function of pH in aqueous solutions prepared using NH3 and NaH13CO3. Apart from the
184 substitution of the 13C-labelled bicarbonate, the spectroscopic conditions were identical to
185 those used to measure CK activity, and the results are illustrated in Figure 6. This shows that
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186 with 0.4 M NH3 and NaH13CO3, the amount of carbamate drops from about 56 mM at pH 9.9,
187 corresponding to 14% of the bicarbonate used, to around 13 mM at pH 11.4, or less than
188 3.5%. Despite this four-fold decrease in carbamate concentration, under the same conditions
189 the activity of each of the hyperthermophilic CKs at pH 11.4 is only less by 15-25%
190 compared to that at pH 9.9.
191 At any given pH, the equilibrium constant (K) of the reactions involved in the formation of D
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192 carbamate (Figure 1) is represented overall by the equation: n
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193 K (dm3 mol-1) = [carbamate]/([NH3 + NH4OH].[CO2 + HCO3-]) ad
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194 K being constant dictates that the carbamate speciation vs pH profile is of the same shape, but f
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195 lower in intensity, with solutions prepared using 0.05 or 0.2, instead of 0.4 M, NH3 and h
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196 NaHCO3 (2, 28, 29). We confirmed this expected relationship using 13C NMR spectroscopy p:/
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197 with 0.2 and 0.4 M NH3 and NaH13CO3. For example, at pH 9.9, the measured carbamate em
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198 concentration of 56 mM with 0.4 M reagents corresponds to K = 0.47 dm3 mol-1. By s
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199 comparison, the carbamate concentration of 17 mM with 0.2 M reagents corresponds to K = r
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200 0.51 dm3 mol-1, the difference being attributable to experimental errors in measurement. n

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201 Similarly, at pH 9.4, the carbamate concentrations were 14 and 50 mM, with 0.2 and 0.4 M ar
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202 reagents, respectively, corresponding to K = 0.40 and 0.41 dm3 mol-1. Although limitations to 2
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203 13C NMR spectroscopy make it difficult to accurately measure the relatively small amounts 20
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204 of carbamate present in solutions with lower reagent concentrations and at the extremes of the b
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205 pH range 7.9-11.4, the values may thus be reliably predicted to follow the shape of the higher u
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206 reagent concentration profile (Figure 6), with the carbamate concentration at pH 11.4 being t
207 less than a quarter of that at pH 9.9. Yet, notwithstanding the lower carbamate concentration,
208 the activity of CK-Pf, CK-Ts, and CK-Tb is as high at pH 11.4 as it is pH 9.9, with solutions
209 prepared using either 0.05 or 0.2 NH3 and NaHCO3. Each of these enzymes much more
210 efficiently processes the available carbamate at the higher pH value.
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211 As a further probe of CK robustness, enzyme behavior was also investigated at various
212 temperatures. The results of representative analyses at pH 9.9, again with CK-Pf, CK-Ts, CK-
213 Tb and CK-Fn, in solutions prepared using 0.2 M NH3 and NaHCO3, at 20, 40, 60 and 80 °C,
214 are presented in Figure 7. Temperature-dependence introduces further complexity into the
215 enzyme kinetics. Even so, all the hyperthermophilic CKs show increasing activity with
216 temperature and maximum catalysis at 80 °C. The thermophilic CK-Fn is most active at D
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217 40 °C, but shows decreased function at higher temperatures. CK-Ts and CK-Tb were found to wn
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218 be the most active at 80 °C, and maintain catalysis over the broadest temperature range. CK- d
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219 Tb generally shows the highest activity of all the CKs tested. It is noteworthy that the activity f
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220 of this enzyme, which normally functions at a physiological temperature above 80 °C, is even
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221 comparable to that of CK-Fn (and CK-Tm and CK-Ef – data not shown) at 20-40 °C. This :
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222 behavior is rare, as few hyperthermophilic enzymes have been found to show similar catalytic m
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223 efficiency to their non-hyperthermophilic counterparts at these lower temperatures (30, 31). s
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224 This property is particularly relevant as lower temperatures may be preferable, or even r
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225 required, for industrial processes utilizing these enzymes. It was also of interest to investigate n

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226 whether the activity of the hyperthermophilic CKs at high pH and temperature correlates with ar
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227 stability over time, an important property in applied biocatalysis. There was no measurable 2
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228 change in catalysis with any of the hyperthermophilic CKs after incubation in buffer at 40 °C 0
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229 for 60 h. By contrast, CK-Fn was inactivated through this treatment. In addition, CK-Pf and b
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230 CK-Tb were stored at 4 °C for six months and still retained full catalytic activity. u
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231 Overall, the three hyperthermophilic CKs that show high sequence similarity, CK-Pf, t
232 CK-Ts and CK-Tb, display high activity at pH 9.4-11.4 and, as mentioned above, are more
233 efficient at processing the available carbamate at the top of this pH range. It is intriguing to
234 note that the CKs that share these properties are all likely to operate physiologically in the
235 anabolic direction. However, based on the available evidence, it is not possible to predict
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