Malate Synthase and β-Methylmalyl Coenzyme A Lyase Reactions in the Methylaspartate Cycle in Haloarcula hispanica

ABSTRACT

Haloarchaea are extremely halophilic heterotrophic microorganisms belonging to the class Halobacteria (Euryarchaeota). Almost half of the haloarchaea possesses the genes coding for enzymes of the methylaspartate cycle, a recently discovered anaplerotic acetate assimilation pathway. In this cycle, the enzymes of the tricarboxylic acid cycle together with the dedicated enzymes of the methylaspartate cycle convert two acetyl coenzyme A (acetyl-CoA) molecules to malate. The methylaspartate cycle involves two reactions catalyzed by homologous enzymes belonging to the CitE-like enzyme superfamily, malyl-CoA lyase/thioesterase (haloarchaeal malate synthase [hMS]; Hah_2476 in Haloarcula hispanica) and β-methylmalyl-CoA lyase (haloarchaeal β-methylmalyl-CoA lyase [hMCL]; Hah_1341). Although both enzymes catalyze the same reactions, hMS was previously proposed to preferentially catalyze the formation of malate from acetyl-CoA and glyoxylate (malate synthase activity) and hMCL was proposed to primarily cleave β-methylmalyl-CoA to propionyl-CoA and glyoxylate. Here we studied the physiological functions of these enzymes during acetate assimilation in H. hispanica by using biochemical assays of the wild type and deletion mutants. Our results reveal that the main physiological function of hMS is malyl-CoA (not malate) formation and that hMCL catalyzes a β-methylmalyl-CoA lyase reaction in vivo. The malyl-CoA thioesterase activities of both enzymes appear to be not essential for growth on acetate. Interestingly, despite the different physiological functions of hMS and hMCL, structural comparisons predict that these two proteins have virtually identical active sites, thus highlighting the need for experimental validation of their catalytic functions. Our results provide further proof of the operation of the methylaspartate cycle and indicate the existence of a distinct, yet-to-be-discovered malyl-CoA thioesterase in haloarchaea.

IMPORTANCE Acetate is one of the most important substances in natural environments. The activated form of acetate, acetyl coenzyme A (acetyl-CoA), is the high-energy intermediate at the crossroads of central metabolism: its oxidation generates energy for the cell, and about a third of all biosynthetic fluxes start directly from acetyl-CoA. Many organic compounds enter the central carbon metabolism via this key molecule. To sustain growth on acetyl-CoA-generating compounds, a dedicated assimilation (anaplerotic) pathway is required. The presence of an anaplerotic pathway is a prerequisite for growth in many environments, being important for environmentally, industrially, and clinically important microorganisms. Here we studied specific reactions of a recently discovered acetate assimilation pathway, the methylaspartate cycle, functioning in extremely halophilic archaea.

INTRODUCTION

Acetyl coenzyme A (acetyl-CoA) is an important intermediate in central carbon metabolism. Many organic compounds, such as alcohols, fatty acids, some amino acids, and polyhydroxyalkanoates, enter the central carbon metabolism via this key molecule. Its further assimilation requires a dedicated anaplerotic pathway. In anaerobes, acetyl-CoA is usually carboxylated to pyruvate by the oxygen-sensitive pyruvate synthase, which uses reduced ferredoxin as an electron donor (1, 2). For (facultative) aerobes, three acetyl-CoA assimilation pathways are known, the glyoxylate cycle, the ethylmalonyl-CoA pathway, and the methylaspartate cycle (3–5). The latter pathway has recently been discovered in Haloarcula marismortui and Haloarcula hispanica and probably functions in almost half of the sequenced haloarchaeal species (5, 6). The other half appears to use the glyoxylate cycle instead (6).

In the methylaspartate cycle, acetyl-CoA and oxaloacetate are converted to glutamate via the reactions of the tricarboxylic acid cycle and glutamate dehydrogenase. The rearrangement of glutamate to methylaspartate and its subsequent deamination lead to mesaconate (methylfumarate), which is then activated to mesaconyl-CoA and hydrated to β-methylmalyl-CoA. The latter is cleaved into propionyl-CoA and glyoxylate. Propionyl-CoA carboxylation leads to succinyl-CoA, which is used to regenerate oxaloacetate, thus closing the cycle (Fig. 1). The glyoxylate that is produced in this process is combined with another molecule of acetyl-CoA, which yields the final methylaspartate cycle assimilation product, malate (5).

FIG 1.

The methylaspartate cycle functioning in haloarchaea (5). The characteristic reactions of the cycle are shown in red, and the names of the corresponding genes and proteins are as follows: mamAB, glutamate mutase; mal, methylaspartate ammonia lyase; mct, mesaconate CoA transferase; mch, mesaconyl-CoA hydratase; mcl, β-methylmalyl-CoA lyase; ms, apparent malate synthase.

Deletion of the genes encoding the enzymes responsible for glutamate conversion to β-methylmalyl-CoA results in loss of the ability of H. hispanica to grow on acetate alone (6). Interestingly, the enzymes responsible for the formation of glyoxylate and propionyl-CoA from β-methylmalyl-CoA (haloarchaeal β-methylmalyl-CoA lyase [hMCL]; Hah_1341) and for the glyoxylate condensation with acetyl-CoA to malate (haloarchaeal malate synthase [hMS]; Hah_2476) are close relatives (37% amino acid sequence identity) within the CitE-like enzyme superfamily (Fig. 2) (5, 7). Thus, they may potentially substitute for each other. Indeed, in the reaction catalyzed by hMS, malyl-CoA is a free intermediate that can easily be detected in the reaction mixture (5). As this compound is not released from the active site and thus is not detectable for canonical malate synthases (i.e., Escherichia coli malate synthase A) (8), hMS may also be called malyl-CoA lyase/thioesterase (5). Interestingly, the characterization of heterologously produced and refolded hMCL and hMS from H. marismortui showed that both enzymes are able to catalyze the same reactions, though with different catalytic efficiencies (5) (Fig. 3). It was proposed that hMS is adapted to catalyze the apparent malate synthase reaction, whereas hMCL is more efficient in cleaving β-methylmalyl-CoA into propionyl-CoA and glyoxylate (5). However, this proposal was based only on the analysis of heterologously produced refolded proteins, which may differ in their catalytic properties from the native enzymes. Refolding efficiency varies for different proteins, and conclusions based on the comparison of catalytic properties of refolded proteins should be considered with caution. For example, the V_max for acetyl-CoA condensation with glyoxylate of hMS is 2 orders of magnitude higher than its V_max for malyl-CoA thioester hydrolysis (Fig. 3). The thioesterase reaction of hMS thus appeared to be a major bottleneck in the methylaspartate cycle and the formation of malate, and we hypothesized that the thioesterase activity of hMCL may contribute to the apparent malate synthase reaction in vivo (5). Furthermore, an additional (specific?) malyl-CoA thioesterase may be present in haloarchaea.

FIG 2.

Maximum-likelihood phylogenetic tree of CitE-like proteins and malate synthases. The haloarchaeal proteins are indicated in red. The crenarchaeal malate synthase cluster consists of homologs of Sulfolobus acidocaldarius malate synthase (34), the Mcl2 cluster consists of homologs of malyl-CoA thioesterase from R. sphaeroides (10), and the citramalyl-CoA lyase cluster consists of homologs of (S)-citramalyl-CoA lyase from Y. pestis, P. aeruginosa, and H. sapiens (16). The functions of other CitE-like proteins are not known.

FIG 3.

Reactions catalyzed by H. marismortui malate synthase and β-methylmalyl-CoA lyase (data are from reference 5). ND*, not determined.

Apart from the methylaspartate cycle, the malate synthase reaction is also involved in the two other acetyl-CoA assimilation pathways, the glyoxylate cycle and the ethylmalonyl-CoA pathway (Fig. 4). In the glyoxylate cycle, enzymes of the tricarboxylic acid cycle together with the first key enzyme of the cycle, isocitrate lyase, oxidize acetyl-CoA to glyoxylate. Malate synthase, the second key enzyme, catalyzes a one-step condensation of acetyl-CoA and glyoxylate to malate, the final product of the glyoxylate cycle (3). Interestingly, like haloarchaea that use the methylaspartate cycle, haloarchaea employing the glyoxylate cycle for acetate assimilation also possess an hMS instead of a classical malate synthase (5). In contrast, in the ethylmalonyl-CoA pathway, two acetyl-CoA molecules are converted to crotonyl-CoA, which is then reductively carboxylated to ethylmalonyl-CoA. The latter is converted in several steps into β-methylmalyl-CoA, which is then cleaved to propionyl-CoA and glyoxylate. Notably, the condensation of glyoxylate and acetyl-CoA to malyl-CoA is catalyzed by the same enzyme that catalyzes the β-methylmalyl-CoA lyase reaction (Mcl1 in Rhodobacter sphaeroides) (Fig. 4) (9), whereas hydrolysis of malyl-CoA to malate and free CoA is catalyzed by a (specific) malyl-CoA thioesterase (Mcl2) homologous to Mcl1 (34% amino acid sequence identity) (Fig. 2) (10).

FIG 4.

Roles of malyl-CoA/β-methylmalyl-CoA lyases and malyl-CoA lyase/thioesterases (red) in different acetate assimilation pathways: the methylaspartate cycle in H. hispanica (A), the glyoxylate cycle in Haloferax volcanii (B), and the ethylmalonyl-CoA pathway in R. sphaeroides (C).

This study aimed to elucidate the physiological functions of hMCL and hMS in H. hispanica by using biochemical assays of the wild type and deletion mutants. Our data show that hMS preferentially catalyzes malyl-CoA formation from acetyl-CoA and glyoxylate in cells. Moreover, hMCL primarily catalyzes the β-methylmalyl-CoA lyase reaction in vivo. However, both enzymes can partially functionally substitute for each other in the corresponding deletion strains and are therefore, each on their own, not essential for growth on acetate. Interestingly, malyl-CoA thioesterase activity was still detectable at comparably high levels in a double deletion strain. Our results provide further proof of the operation of the methylaspartate cycle and indicate the existence of a (novel) distinct, yet-to-be-discovered malyl-CoA thioesterase in haloarchaea.

RESULTS

H. hispanica wild-type cells were grown in medium with acetate as the sole carbon source with a generation time of 30 h corresponding to a specific growth rate of 0.023 h⁻¹ and a specific carbon assimilation rate of 30 nmol min⁻¹ mg of protein⁻¹. Pyruvate-grown cells were used as a control. To study the physiological functions of hMS and hMCL, the corresponding Δmcl (Δhah_1341), malate synthase gene deletion (ΔMS [Δhah_2476]), and Δmcl ΔMS (Δhah_1341 Δhah_2476) mutants were constructed. The growth of the Δmcl and ΔMS mutant strains in medium with acetate as the sole carbon source was impaired, whereas the double mutant was not able to grow in this medium at all. In contrast, growth on pyruvate was not affected (Fig. 5). These data suggest that hMCL and hMS can partially functionally substitute for each other, thus being able to catalyze all of the required reactions. Furthermore, the growth phenotype of the deletion mutants indicates the involvement of these enzymes in acetate assimilation in H. hispanica, in agreement with their proposed role in the methylaspartate cycle (5). Notably, the Δmcl strain grew with a longer lag phase on acetate than the ΔMS strain (6 and 2 days, respectively), whereas the growth rates of these two strains were almost the same (0.016 and 0.015 h⁻¹, respectively) (Fig. 5).

FIG 5.

Growth of H. hispanica wild-type strain ATCC 33960 (A) and Δmcl (Δhah_1341) (B), ΔMS (Δhah_2476) (C), and Δmcl ΔMS (Δhah_1341 Δhah_2476) (D) mutant strains on medium with acetate (■) or pyruvate (●). The experiment was performed in triplicate. The error bars represent standard deviations. OD₅₇₈, optical density at 578 nm.

In accordance with the growth experiments, apparent malate synthase activity (glyoxylate-dependent CoA formation from acetyl-CoA) was found in cell extracts of the wild type, as well as in the ΔMS and Δmcl mutant strains grown on acetate (Table 1). However, the intermediary formation of considerable amounts of malyl-CoA was detected only in cell extracts of the Δmcl strain (Fig. 6). It could be explained by elevated malyl-CoA lyase activity in this strain (Table 1; for the discussion, see below). The activity of β-methylmalyl-CoA lyase was an order of magnitude lower in the Δmcl strain than in the wild type, though only a small change (<2-fold) in β-methylmalyl-CoA lyase activity was observed in the ΔMS mutant (Table 1). Notably, both malate synthase and β-methylmalyl-CoA lyase activities are significantly downregulated in pyruvate-grown cells and completely absent from the double mutant (Table 1), thus evidencing the absence of other enzymes with malate synthase or malyl-CoA/β-methylmalyl-CoA lyase activity in H. hispanica. For comparison, the activities of constitutively expressed isocitrate dehydrogenase (involved in the tricarboxylic acid cycle) and of acetate-induced mesaconyl-CoA hydratase (from the methylaspartate cycle) (5, 6) were measured (Table 1). As expected, isocitrate dehydrogenase activity was not regulated, whereas mesaconyl-CoA hydratase activity was present in acetate-grown cells but not in pyruvate-grown cells (as long as pyruvate-grown cells were not incubated with acetate, as was done with the double mutant, which does not grow on acetate alone).

TABLE 1.

Specific activities of various carbon metabolism enzymes in cell extracts of acetate- and pyruvate-grown H. hispanica wild-type ATCC 33960, acetate-grown Δhah_2476 and Δhah_1341 mutant strains, and a pyruvate-grown Δhah_1341 Δhah_2476 double mutant strain^a

Enzyme and method	Sp act (nmol min−1 mg of protein−1) for:
	WT on acetate	WT on pyruvate	Δmcl (Δhah_1341) mutant	ΔMS (Δhah_2476) mutant	Δmcl ΔMS (Δhah_1341 Δhah_2476) mutant
(S)-Malyl-CoA lyase
Malyl-CoA formation, UPLC	19 ± 6	29 ± 13	149 ± 9	15 ± 4	<0.1
Malyl-CoA cleavage, UPLC	13 ± 2	10 ± 5	42 ± 5	7 ± 1	<0.1
Malate synthaseb
CoA formation, UPLC	72 ± 7	23 ± 7	86 ± 23	34 ± 18	<0.1
CoA formation, photometric	41 ± 4	7 ± 3	36 ± 8	23 ± 3	<0.1
β-Methylmalyl-CoA lyase
β-Methylmalyl-CoA cleavage	22 ± 3	1.3 ± 0.7	3 ± 2	12 ± 4	<0.1
β-MethylMalyl-CoA formation	160 ± 2	2.8 ± 1.4	16 ± 8	200 ± 110	<1
β-Methylmalyl-CoA thioesterase, CoA formation, UPLC	<0.1	<0.1	<0.1	<0.1	<0.1
(S)-Malyl-CoA thioesterase, CoA formation, UPLC	107 ± 15	66 ± 33	72 ± 8	92 ± 24	51 ± 12
Isocitrate dehydrogenase	640 ± 60	440 ± 30	490 ± 90	600 ± 160	370 ± 50
Mesaconyl-CoA hydratase	130 ± 30	<0.1	240 ± 90	500 ± 20	130 ± 10

^aAs the double mutant was not able to grow on acetate as a sole carbon source, pyruvate-grown cells were resuspended in acetate medium and incubated for 4 days. The data are from at least three biological replicates. WT, wild type.

^bThe activities in UPLC-based assays were higher than those measured spectrophotometrically. This is not surprising, as the acetyl-CoA concentrations were different in these two assays (1.0 and 0.2 mM). Note that the K_m values of hMS and hMCL for acetyl-CoA were <1 mM (Fig. 3) (5 and 35).

FIG 6.

Time course of acetyl-CoA and glyoxylate condensation reaction catalyzed by cell extracts of wild-type strain ATCC 33960 (A) and ΔMS (Δhah_2476) (B) and Δmcl (Δhah_1341) (C) mutant strains. Symbols: ■, acetyl-CoA; ●, malyl-CoA; ◆, free CoA. The protein concentrations in experiments A to C were 4.6, 2.4, and 5.8 mg ml⁻¹, respectively. The experiments were done in duplicate; the results of typical experiments are shown. Conc., concentration.

Interestingly, malyl-CoA thioesterase activity was scarcely affected in the mutants studied and present also in pyruvate-grown cells (Table 1), thus being virtually independent from both hMS and hMCL. Furthermore, malyl-CoA thioesterase was probably a bottleneck for the apparent malate synthase activity in the Δmcl strain (but not for growth of the Δmcl strain), as the rate of malyl-CoA formation from acetyl-CoA and glyoxylate in the Δmcl strain was higher than in all of the other strains tested (Table 1). This not-yet-identified thioesterase seems to be highly specific for malyl-CoA; no β-methylmalyl-CoA thioesterase activity was detected in H. hispanica. Its K_m toward malyl-CoA was between 50 and 100 μM, as determined by measurements in cell extracts of the wild-type and mutant strains (Table 2), a value that is similar to that of malyl-CoA thioesterase Mcl2 from Rhodobacter sphaeroides (0.09 mM) (10).

TABLE 2.

Apparent K_m and V_max values for malyl-CoA lyase and malyl-CoA thioesterase reactions in cell extracts of H. hispanica wild-type ATCC 33960 and Δhah_2476 (ΔMS), Δhah_1341 (Δmcl), and Δhah_1341 Δhah_2476 (Δmcl ΔMS) mutant strains grown on different substrates^a

Strain, growth conditions	Mean Km (mM), Vmax (nmol min−1 mg of protein−1) ± SD
	Malyl-CoA lyase, reversed acetyl-CoA + glyoxylate → (S)-malyl-CoA	Malyl-CoA thioesterase (S)-malyl-CoA → malate + CoA
WT,b acetate	43 ± 6, 350 ± 20	0.06 ± 0.01, 150 ± 6
WT, pyruvate	NDc	0.08 ± 0.02, 80 ± 5
Δhah_1341 (Δmcl) mutant, acetate	5.8 ± 1.8, 450 ± 40	0.09 ± 0.02, 220 ± 16
Δhah_2476 (ΔMS) mutant, acetate	160 ± 90, 590 ± 220	0.06 ± 0.01, 117 ± 6
Δhah_1341 Δhah_2476 (Δmcl ΔMS) mutant, pyruvate, acetate	NAd	0.08 ± 0.02, 70 ± 6
Δhah_1341 Δhah_2476 (Δmcl ΔMS) mutant, pyruvate	ND	0.10 ± 0.02, 67 ± 4

^aThe values shown for the lyase reaction are for glyoxylate. The acetyl-CoA concentration in the assay mixture was 1 mM and thus higher than the hMS and hMCL K_m values for acetyl-CoA (Fig. 3) (5 and 35). As the double mutant was not able to grow on acetate as a sole carbon source, pyruvate-grown cells were resuspended in acetate medium and incubated for 4 days (these conditions are indicated as pyruvate, acetate). The data are from at least three biological replicates.

^bWT, wild type.

^cND, not determined.

^dNA, no activity.

The presence of an additional malyl-CoA thioesterase in H. hispanica indicates that hMS and hMCL function as malyl-CoA and/or β-methylmalyl-CoA lyases in vivo, while their thioesterase activities appear to be not crucial for the operation of the methylaspartate cycle. To better understand the differences between hMS and hMCL, we studied the K_m value of the (S)-malyl-CoA lyase reaction for glyoxylate in the ΔMS and Δmcl mutant strains (Table 2). Although the K_m value in the Δmcl mutant was close to the K_m values of the characterized MCLs from Chloroflexus aurantiacus and Rhodobacter sphaeroides (2.0 and 3.1 mM, respectively; 10, 11), the K_m in the ΔMS mutant was extremely high (160 mM). The apparent K_m in wild-type cells was found to be between these two extremes.

On the basis of the enzyme kinetics in the cell extracts of mutants (Table 1), including the apparent K_m values of the enzymes for glyoxylate in vivo (Table 2), we conclude that the physiological functions of hMCL (Hah_1341) and hMS (Hah_2476) are β-methylmalyl-CoA cleavage (to propionyl-CoA and glyoxylate) and malyl-CoA synthesis (from acetyl-CoA and glyoxylate), respectively. The results of the determination of mesaconate concentrations in H. hispanica cells confirmed the proposed function of these enzymes. We have previously shown that this intermediate of the methylaspartate cycle accumulates in the cells if downstream reactions of the cycle are impaired (6). Although mesaconate was not detected in the wild-type and ΔMS mutant strains (concentration of <1 mM in cytoplasm), the Δmcl strain and the double mutant accumulated large amounts of this compound (33 ± 13 and 129 ± 41 mM, respectively). Thus, the disruption of mcl (but not of the MS gene) blocks further metabolism of mesaconate and leads to its accumulation, as expected for the functional methylaspartate cycle (Fig. 1).

To study the properties of hMS functioning in the context of the glyoxylate cycle, we grew Haloferax mediterranei on acetate and studied cell extracts for the corresponding activities. As expected of an archaeon containing hMS (6, 12), we could measure high malyl-CoA and β-methylmalyl-CoA lyase, as well as malyl-CoA thioesterase, activities but almost no hydrolysis of β-methylmalyl-CoA (Table 3). These data show that hMSs from Haloarcula and Haloferax have similar properties, despite functioning in different organisms and participating in different metabolic pathways.

TABLE 3.

Specific activities of carbon metabolism enzymes in cell extracts of Haloferax mediterranei grown in acetate or rich medium^a

Enzyme (method)	Mean sp act (nmol min−1 mg of protein−1) ± SD
	Acetate	Rich medium
Malate synthase (CoA formation)	1,100 ± 70	25 ± 12
(S)-Malyl-CoA lyase (malyl-CoA formation)	1,830 ± 520	25 ± 12
Malyl-CoA thioesterase (CoA formation)	1,170 ± 40	48 ± 24
β-Methylmalyl-CoA thioesterase (CoA formation)	17 ± 14	1.4 ± 0.7
β-Methylmalyl-CoA lyase (β-methylmalyl-CoA cleavage)	600 ± 320	<0.1

^aThe data are from three biological replicates. All enzymes were measured with UPLC-based assays.

DISCUSSION

CitE superfamily proteins and homologous malate synthases catalyze two types of reactions, an irreversible CoA ester hydrolysis and/or a reversible C-C bond cleavage (aldol reaction) (7). Interestingly, all known thioesterases of this enzyme family are highly specific for malyl-CoA, whereas the C-C bond lyases are often promiscuous. For example, hMCL and hMS catalyze malyl-CoA ester hydrolysis but also the C-C bond cleavage with malyl-CoA, β-methylmalyl-CoA, and (S)-citramalyl-CoA (5). The inability of these enzymes to hydrolyze the closely related substrate β-methylmalyl-CoA is remarkable and suggests their specific adaptation to function in the methylaspartate cycle, as this substrate binds to the same active site during the catalysis of the β-methylmalyl-CoA lyase reaction (7, 13). The hydrolysis of β-methylmalyl-CoA would lead to the formation of a dead-end product (β-methylmalate) and is therefore undesired. Interestingly, although it is possible to predict the respective substrate and reaction specificities for enzymes of the CitE superfamily by amino acid sequence alignment and phylogenetic analysis (Fig. 2) (7), the active-site architectures (residues and relative distances) of all of these enzymes are virtually identical (Fig. 7). Why hMCL prefers propionyl-CoA or β-methylmalyl-CoA as a substrate, respectively, whereas hMS prefers acetyl-CoA or malyl-CoA (Fig. 3; Table 1) is not obvious from active-site residue comparisons (Fig. 7). The residues closest to the terminal methyl group of propionyl-CoA are completely conserved among canonical and haloarchaeal malate synthases, as well as bacterial and haloarchaeal malyl-CoA or β-methylmalyl-CoA lyases. Furthermore, the binding of glyoxylate is achieved by coordination with a divalent ion (typically Mg²⁺) and hydrogen bonding with surrounding residues (7, 13), which are also completely conserved among these enzymes. The differences in orders of magnitude of the K_m values for glyoxylate are thus quite intriguing. Finally, why some enzymes are able to hydrolyze the CoA thioester bond of specifically malyl-CoA and not β-methylmalyl-CoA is not understood. Notably, haloarchaea that possess the glyoxylate cycle do not synthesize β-methylmalyl-CoA in their metabolism (e.g., Haloferax spp.). Thus, their hMS does not experience evolutionary pressure to avoid β-methylmalyl-CoA hydrolysis. Still, Haloferax hMS has only very low β-methylmalyl-CoA thioesterase activity (Table 3). This striking preference for malyl-CoA over β-methylmalyl-CoA suggests that the enzyme probably evolved to function in the methylaspartate cycle and retained its original substrate specificity in Haloferax. This may be regarded as an indication that the methylaspartate cycle is an ancestral feature of haloarchaea and that the glyoxylate cycle emerged in this group later, probably after acquisition of the isocitrate lyase gene by lateral gene transfer (14).

FIG 7.

Superposition of active-site residues of the hMS of H. volcanii (light pink; PDB 3OYZ), the malyl-CoA/β-methylmalyl-CoA/citramalyl-CoA lyase of C. aurantiacus (light blue; PDB 4L80), the malyl-CoA/β-methylmalyl-CoA of R. sphaeroides (dark blue; PDB 4L9Y), a homology model of the hMCL from H. hispanica (teal), and a homology model of the hMS from H. hispanica (dark pink). The numbering of residues corresponds to the hMS of H. volcanii, which has Mg²⁺, pyruvate (PYR), and acetyl-CoA (ACO) bound. The MCL from C. aurantiacus has propionyl-CoA and oxalate bound instead. Coordination of Mg²⁺ by two water molecules, pyruvate, and active-site residues is outlined for the hMS of H. volcanii.

Among the substrates for the C-C bond lyase reaction of hMS and hMCL, (S)-citramalyl-CoA does not appear to be physiologically relevant. The activity of (S)-citramalyl-CoA lyase is probably a result of a high similarity of citramalyl-CoA to β-methylmalyl-CoA. However, all three reactions are involved in the 3-hydroxypropionate bi-cycle in Chloroflexus aurantiacus and are catalyzed by the same enzyme (7, 11, 15). The Chloroflexus enzyme catalyzes these reactions in reversed directions compared to the haloarchaeal enzymes, as functioning of the 3-hydroxypropionate bi-cycle requires malyl-CoA and citramalyl-CoA cleavage but β-methylmalyl-CoA formation (11, 15). The Chloroflexus malyl-CoA/β-methylmalyl-CoA/citramalyl-CoA lyase does not catalyze a thioesterase reaction, which would be undesirable in the 3-hydroxypropionate bi-cycle (7, 11).

The CitE proteins involved in itaconate assimilation/detoxification as (S)-citramalyl-CoA lyases form a separate branch of the phylogenetic tree (Fig. 2). They are either highly specific to their substrate, (S)-citramalyl-CoA (Yersinia pestis), or catalyze (S)-citramalyl-CoA and (R)-3-hydroxy-3-methylglutaryl-CoA lyase reactions (Pseudomonas aeruginosa and Homo sapiens enzymes) (16). Although low malate and β-methylmalate synthase activities had been demonstrated for H. sapiens CitE (17), these enzymes appear to be dedicated lyases that barely catalyze thioesterase reactions (16).

Using cell extracts of acetate-grown Δmcl and ΔMS mutant strains of H. hispanica, we determined the K_m values of hMS and hMCL for glyoxylate. They were surprisingly different not only from each other (6 and 160 mM, respectively) (Table 2) but also from the values measured with heterologously produced and reconstituted enzymes from H. marismortui (0.5 and 27 mM) (Fig. 3). As it is unexpected that heterologously produced and reconstituted enzymes will have lower (i.e., better) K_m values than native proteins, we attribute these results to differences in the proteins of different species. Since K_m values reflect intracellular metabolite concentrations (18), it appears that glyoxylate is present in H. hispanica cells at millimolar levels during growth on acetate. Nevertheless, the high apparent K_m value of hMCL for glyoxylate during malyl-CoA formation in the ΔMS strains may again hint at a preference for propionyl-CoA over acetyl-CoA. The K_m value for glyoxylate seems to be dependent on the CoA thioester in H. marismortui as well (Fig. 3). Similarly, the 1 order of magnitude faster malyl-CoA formation in the Δmcl strain may point at overexpression of hMS in order to maintain some level of β-methylmalyl-CoA cleavage activity, which is not the preferred reaction catalyzed by hMS.

Our data suggest the presence of an additional malyl-CoA thioesterase in H. hispanica. This thioesterase may not be a member of the CitE enzyme superfamily. However, the H. hispanica genome contains an additional citE-like gene of unknown function. Nevertheless, our phylogenetic analysis suggests that this protein is closely related to citramalyl-CoA lyases and therefore catalyzes a C-C bond lyase rather than a thioesterase reaction (Fig. 2). Malyl-CoA thioesterase activity could be catalyzed by a specific enzyme or be a result of promiscuity of one of the H. hispanica enzymes involved in central metabolism and synthesized constitutively.

In summary, the results of this study prove the functioning of Hah_1341 as β-methylmalyl-CoA lyase and of Hah_2476 as malyl-CoA forming malyl-CoA lyase in the methylaspartate cycle in H. hispanica and suggest the existence of an additional malyl-CoA thioesterase in this microorganism. These data provide further support for the functioning of the methylaspartate cycle in haloarchaea and extend our knowledge of alternative acetate assimilation pathways.

MATERIALS AND METHODS

Microbial strains and culture conditions.

H. hispanica strain ATCC 33960 was grown under aerobic conditions at pH 7.2 to 7.5 and 37°C in a 10-liter flask with an air pump or in shaken 2-liter flasks on a chemically defined medium (19) with acetate (0.2%, wt/vol) or pyruvate (0.2%, wt/vol) as a carbon source. For the cultivation of uracil auxotrophic (pyrF deleted) strain DF60 (20) and its derivatives, uracil (stock solution of 50 mg ml⁻¹ dissolved in dimethyl sulfoxide) was added at a concentration of 50 mg liter⁻¹. Haloferax mediterranei strain ATCC 33500 was also grown under aerobic conditions at pH 7.2 to 7.5 and 37°C in shaken 2-liter flasks on nutrient-rich AS-168 medium (20, 21) or a chemically defined medium (19) with acetate (0.2%, wt/vol).

Materials.

Chemicals were obtained from Fluka (Neu-Ulm, Germany), Sigma-Aldrich (Deisenhofen, Germany), Merck (Darmstadt, Germany), Serva (Heidelberg, Germany), or Roth (Karlsruhe, Germany). Biochemicals were from Roche Diagnostics (Mannheim, Germany), AppliChem (Darmstadt, Germany), or Gerbu (Craiberg, Germany). Materials for cloning were purchased from New England BioLabs (Frankfurt, Germany), Novagen (Schwalbach, Germany), Genaxxon Bioscience GmbH (Biberach, Germany), MWG Biotech AG (Ebersberg, Germany), Biomers (Ulm, Germany), or Qiagen (Hilden, Germany). Primers were synthesized by Thermo Fisher Scientific Invitrogen (Beijing, China).

Synthesis.

Acetyl-CoA was synthesized from its anhydride by the method of Simon and Shemin (22). β-Methylmalyl-CoA and (S)-malyl-CoA were synthesized enzymatically with recombinant (S)-malyl-CoA/β-methylmalyl-CoA/(S)-citramalyl-CoA lyase from Chloroflexus aurantiacus (15) as described previously (16). A mixture of mesaconyl-C1-CoA and mesaconyl-C4-CoA was synthesized chemically from the free acid by the mixed-anhydride method of Stadtman (23). From this mixture, mesaconyl-C1-CoA was purified by high-performance liquid chromatography (15). The dry powders of the CoA esters were stored at −20°C.

Mutant construction and verification.

The strains, plasmids, and primers used for mutant construction and verification are listed in Tables S1 and S2 in the supplemental material. Mutant construction was performed by the pop-in/pop-out method described previously (20). The gene deletion mutants were selected by PCR verification and confirmed by sequence analysis. The transformation of H. hispanica was performed by the polyethylene glycol-mediated method (24).

Preparation of cell extracts.

Cell extracts were prepared under oxic conditions with an ultrasonic homogenizer Bandelin UW mini20 (BANDELIN Electronic GmbH & Co., Berlin, Germany). Cells (100 mg) were suspended in 0.2 ml of 50 mM Tris HCl, pH 7.8, containing 2 M KCl and 0.1 mg ml⁻¹ DNase I in 1.5-ml Eppendorf vials. Cell suspensions were sonicated for 3 × 60 s (0.5-s pulses, 70% power) on ice and then centrifuged (14,000 × g, 4°C, 20 min), and the supernatants (cell extracts) were used for enzyme assays. The protein content of the cell extract was 2 to 12 mg ml⁻¹.

Enzyme assays.

Spectrophotometric enzyme assays (0.5-ml assay mixture) were performed aerobically in 0.5-ml cuvettes at 37°C. Reactions involving NAD(P)H were measured at 365 nm (ε_NADH = 3.4 mM⁻¹ cm⁻¹, ε_NADPH = 3.5 mM⁻¹ cm⁻¹) (25). Reactions with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) were measured at 412 nm (ε_DTNB-CoA = 14.2 mM⁻¹ cm⁻¹) (26).

Isocitrate dehydrogenase activity was measured as isocitrate-dependent reduction of NAD(P)⁺ in a reaction mixture containing 100 mM Tris HCl (pH 7.8), 2 M KCl, 5 mM MgCl₂, 1 mM NAD(P)⁺, 4 mM dl-isocitrate, and cell extract (20 to 60 μg of protein). The activity was found only with NADP⁺ as a cofactor.

Malate synthase activity was detected by two methods: spectrophotometrically as glyoxylate-dependent release of CoA from acetyl-CoA with DTNB and by ultraperformance liquid chromatography (UPLC) as glyoxylate-dependent free CoA formation from acetyl-CoA. The reaction mixture for the spectrophotometric assay contained 100 mM Tris HCl (pH 7.8), 3 M KCl, 5 mM MgCl₂, 1 mM DTNB, 0.2 mM acetyl-CoA, 4 mM glyoxylate, and cell extract (40 to 120 μg of protein). For the UPLC-based assay, the reaction mixture (25 μl) contained 100 mM Tris HCl (pH 7.8), 3 M KCl, 5 mM MgCl₂, 10 mM glyoxylate, 1 mM acetyl-CoA, and cell extract (5 to 30 μg of protein). The reaction was started by the addition of acetyl-CoA. After appropriate time intervals, the reaction was stopped by transfer to ice and the addition of 10 μl of 2 M HCl–10% acetonitrile. Protein was removed by centrifugation, and the samples were analyzed by reverse-phase (RP) C₁₈ UPLC as described previously (16).

(S)-Malyl-CoA lyase was measured either by the formation of (S)-malyl-CoA from acetyl-CoA and glyoxylate or by the formation of acetyl-CoA from (S)-malyl-CoA. For measurement in the direction of malyl-CoA formation, the reaction mixture (25 μl) contained 100 mM Tris HCl (pH 7.8), 3 M KCl, 5 mM MgCl₂, 10 mM glyoxylate, 1 mM acetyl-CoA, and cell extract (5 to 30 μg of protein). For measurement in the direction of malyl-CoA cleavage, the reaction mixture (25 μl) contained 100 mM morpholinepropanesulfonic acid (MOPS)-KOH (pH 7.0), 3 M KCl, 5 mM MgCl₂, 0.5 mM (S)-malyl-CoA, 3.5 mM phenylhydrazine, and cell extract (5 to 30 μg of protein). The reaction was started by the addition of CoA ester and stopped after appropriate time intervals as described above. The products were analyzed by RP C₁₈ UPLC.

β-Methylmalyl-CoA lyase was measured in the same manner as described above for malyl-CoA lyase, but (S)-malyl-CoA was replaced with β-methylmalyl-CoA (0.5 mM) and acetyl-CoA was replaced with propionyl-CoA (1 mM).

(S)-Malyl-CoA and β-methylmalyl-CoA thioesterase activities were measured in a UPLC-based assay. The reaction mixture (25 μl) contained 100 mM MOPS-KOH (pH 7.0), 3 M KCl, 5 mM MgCl₂, 0.5 mM (S)-malyl-CoA or β-methylmalyl-CoA, and cell extract (5 to 20 μg of protein). The reaction was started by the addition of CoA ester and stopped after appropriate time intervals as described above. The products were analyzed by RP C₁₈ UPLC.

Determination of K_m and V_max for (S)-malyl-CoA lyase and (S)-malyl-CoA thioesterase in cell extracts of H. hispanica.

K_m values of (S)-malyl-CoA lyase for glyoxylate were measured in a reaction mixture (25 μl) containing 100 mM Tris HCl (pH 7.8), 3 M KCl, 5 mM MgCl₂, 0.1 to 50 mM glyoxylate (1 to 200 mM for the ΔMS mutant), 1 mM acetyl-CoA, and cell extract (4 to 10 μg of protein). For the (S)-malyl-CoA thioesterase reaction, the assay mixture (25 μl) contained 100 mM MOPS-KOH (pH 7.0), 3 M KCl, 5 mM MgCl₂, 0.05 to 1 mM (S)-malyl-CoA, and cell extract (4 to 10 μg of protein). The reaction was started by the addition of CoA ester and stopped after 2 min, as described above. The products were analyzed by RP C₁₈ UPLC.

Database search and phylogenetic analysis.

Query sequences were obtained from the National Center for Biotechnology Information (NCBI) database. BLAST searches were performed via the NCBI BLAST server (http://www.ncbi.nlm.nih.gov/BLAST/) (27). Amino acid sequences were aligned with sequences from GenBank by using CLUSTAL W (28) implemented within BioEdit software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). The phylogenetic tree was reconstructed by using a maximum-likelihood algorithm (29) in MEGA6 (30). One thousand bootstrap replications were conducted to evaluate the reliability of the reconstructed trees. The GenBank accession numbers for the CitE protein sequences are listed in Table S3.

Structural analysis.

Homology modeling and secondary structure predictions were performed by using the Phyre2 online server (31), and Swiss-Model (32). The models where analyzed by using PyMOL (https://www.pymol.org). Multiple templates were used and manually checked for quality before models were chosen for subsequent analysis. The final homology model for the H. hispanica malate synthase was based on PDB 3OYZ (13) as the template, whereas the final model for the H. hispanica β-methylmalyl-CoA lyase was based on PDB 4L9Y (7).

Other methods.

Mesaconate concentrations were determined as described previously (6). CoA and CoA esters were identified and quantified by UPLC with an RP C₁₈ column (BEH C₁₈, 1.7 μm, 2.1 by 100 mm; Waters) as described in reference 16. Protein was measured by the Bradford method (33) with bovine serum albumin as the standard. DNA sequence determination was performed by GATC Biotech (Constance, Germany) and Thermo Fisher Scientific-Invitrogen (Beijing, China).