Activation of a [NiFe]-hydrogenase-4 isoenzyme by maturation proteases.

Maturation of [NiFe]-hydrogenases often involves specific proteases responsible for cleavage of the catalytic subunits. Escherichia coli HycI is the protease dedicated to maturation of the Hydrogenase-3 isoenzyme, a component of formate hydrogenlyase-1. In this work, it is demonstrated that a Pectobacterium atrosepticum HycI homologue, HyfK, is required for hydrogenase-4 activity, a component of formate hydrogenlyase-2, in that bacterium. The P. atrosepticum ΔhyfK mutant phenotype could be rescued by either P. atrosepticum hyfK or E. coli hycI on a plasmid. Conversely, an E. coli ΔhycI mutant was complemented by either E. coli hycI or P. atrosepticum hyfK in trans. E. coli is a rare example of a bacterium containing both hydrogenase-3 and hydrogenase-4, however the operon encoding hydrogenase-4 has no maturation protease gene. This work suggests HycI should be sufficient for maturation of both E. coli formate hydrogenlyases, however no formate hydrogenlyase-2 activity was detected in any E. coli strains tested here.

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Hydrogenases are enzymes that are widespread in microbial systems where they catalyse the oxidation or production of molecular hydrogen (H2) [1]. A major class of hydrogenases common in Proteobacteria are the [NiFe]-hydrogenases that rely on an elaborate Ni-Fe-CO-2CN- metallocofactor at their active sites [1]. These two-part enzymes, consisting of a large subunit (~60 kDa) harbouring the [NiFe]-cofactor and a small subunit (~30 kDa) that contains iron-sulfur clusters, require the coordination of both specific and housekeeping biosynthetic pathways for their assembly and activation [2]. The biosynthesis pathway of the large subunit includes the critical final steps of cofactor assembly and insertion. Here, the HypA and HypB accessory proteins insert the nickel ion in to the large subunit as the final component of the [NiFe]-cofactor, where the HypA monomer interacts with the unstructured N-terminus and a C-terminal beta strand of the immature large subunit [3]. This novel interaction brings the HypA nickel binding site and immature hydrogenase large subunit active site in proximity to allow nickel transfer [3]. Next, and for the vast majority [NiFe]-hydrogenases, one final maturation step is required before small subunit docking and full enzymatic activation can occur. This is the proteolytic cleavage of a short stretch of polypeptide from the C-terminus of the hydrogenase large subunit [4].

Proteolytic maturation of hydrogenases is well understood for the model [NiFe]-hydrogenase-3 isoenzyme (Hyd-3). Here, the Hyd-3 large subunit (encoded by the hycE gene) is cleaved after residue Arg-537 by a specific metallopeptidase termed HycI [5-8]. Removal of the 32-residue C-terminal ‘assembly peptide’ from HycE results in essentially irreversible cofactor-loading, correct folding of the large subunit and successful docking of the small subunit to generate the final active Hyd-3 [2]. Deletion of the hycI gene in led to the complete loss of all Hyd-3 activity and accumulation of an immature, unprocessed version of HycE [9]. Subsequent research in numerous other biological systems resulted in the central dogma that, where [NiFe]-hydrogenase large subunits were synthesized with a C-terminal extension or assembly peptide, that they would require processing by a specific protease for activation, and that said protease would be encoded close to the gene for the large subunit, and that said protease would not normally recognize any other hydrogenase homologues [4].

Hyd-3 is a member of the group 4A [NiFe]-hydrogenases [10] and a component of the formate hydrogenlyase-1 (FHL-1) complex [11]. It is encoded within the hycABCDEFGHI operon that includes the gene for the protease [6, 12]. Interestingly, laboratory strains of encode two separate group 4A [NiFe]-hydrogenases, each predicted to be part of distinct formate hydrogenlyase complexes. Thus in addition to Hyd-3, the [NiFe]-hydrogenase-4 isoenzyme (Hyd-4) is encoded by the hyfABCDEFGHIJR-focB operon [13] and is predicted to be a component of a formate hydrogenlyase-2 (FHL-2) complex [11]. FHL-1 and FHL-2 share the same core architecture, with FHL-2 predicted to contain extra membrane-embedded components [11]. FHL activity is normally maximal under fermentative conditions when the enzyme catalyses the oxidation of formic acid and couples this directly to the reduction of protons to molecular H2. Thus group 4A [NiFe]-hydrogenases have a physiological role in the evolution of hydrogen gas [11]. Directly demonstrating the enzymatic activity of FHL-2 or Hyd-4 has proven challenging. Under laboratory conditions, the enzyme appeared to be neither transcribed nor enzymatically active [14-16], although there is some evidence for a physiological role in H2 metabolism under some specific environmental conditions [17, 18]. In addition, disruption of Hyd-4 genes alone did not affect overall H2 production by [19, 20], again indicating that cellular Hyd-4 activity was very low or absent under the conditions tested. It is also clear that the E. coli hyfABCDEFGHIJR-focB operon does not encode any homologue of HycI (Fig. 1a) nor any other protease [2, 13]. Therefore, while the large subunit of Hyd-4 (HyfG) shares a high degree of sequence identity with HycE including the presence of a 32-residue C-terminal assembly peptide (Fig. 1b), it must also be considered that the apparent low activity of Hyd-4 may stem from incomplete maturation of the enzyme.

Fig. 1.

The genetics of processing group 4A [NiFe]-hydrogenases (a) Schematic showing the genetic organization of K-12 hyc and hyf operons as well as SCRI1043 hyf operon. Gene products are indicated in the legend and colour coded. (Top) The K-12 hycABCDEFGHI operon comprises genes ECK2720-ECK2712. (Middle) The K-12 hyfABCDEFGHIJRfocB operon comprises genes ECK2477-ECK2488. (Bottom) The P. atrospecticum (formerly ) hyfABCDEFGHIJK-hyfR cluster comprises genes ECA1247-ECA1236. The hycI- or hyfK-like genes are highlighted in red. (b) A sequence alignment of the final 93 amino acids, including the proteolytically processed assembly peptides, of HycE, HyfG and HyfG with black to white shading showing most to least conserved residue positions. Sequence alignment was performed using Clustal [45] and presented using BOXSHADE (http://sourceforge.net/projects/boxshade/). (c) The hydrogenase hybrid ('HYD-BRID') C-terminal sequence of the ϕHyfG-HycE construct introduced into FTE001, FTE002, FTE004 and FTE007 strains (Table 1). The sequence coloured black corresponds to the relevant part of the HyfG protein, and the sequence shaded green is the terminal arginine of the mature enzyme and the C-terminal assembly peptide of HycE. (d) A phylogenetic tree of all group 4A [NiFe]-hydrogenase-associated endopeptidases. Homologues were identified using blast [46] before multiple sequence alignment was carried out in Jalview [47]. Phylogenetic trees were constructed using FigTree (http://tree.bio.ed.ac.uk/software/figtree/). Dark blue and yellow colouring highlight those organisms with maturation protease genes associated with hyf and hyc type group 4A subtypes, respectively. Purple and cyan colouring shows organisms with an additional removal or variation in position of the hyfD gene, respectively (occurs within the hyf type only). Red colouring highlights organisms with both hyc and hyf operons (but note that these harbour only one maturation protease gene within their hyc operons). Note that this sequence analysis identified a HyfK homologue in Trabulsiella guaensis, which produces a functional Hyd-4 [29].

In this work, we set out to test the initial hypotheses that the E. coli hyf operon is not sufficiently expressed, and that HyfG is not correctly processed, such that a hydrogenase-null phenotype is observed. To do this we took a recombineering approach and constructed 15 new strains (Table 1 and Supplementary Material, available in the online version of this article) with alternative promoters and/or ϕhyfG::hycE fusion alleles at the native hyf locus on the chromosome. None of the new strains displayed any Hyd-4 activity (Table 1). Briefly, a group of strains with modified hyf transcriptional promoter regions were generated using P1 phage transduction [21] and allelic exchange [22]. All engineering was carried out in single copy on the chromosome, and the strains’ ability to produce H2 gas under fermentative conditions was assayed by gas chromatography [23]. Initially, an K-12 strain (MG056G1, Table 1) was constructed based on the MG1655 parent strain [24] but encoding an internal 10-His tag between residues Gly-85 and Ala-86 within the HyfG protein. The rationale here was that a similarly modified version of HycE (Hyd-3) had retained full activity [25] and that the tag would allow further characterization of Hyd-4 at the protein level if the promoter engineering were successful. Next, the MG056G1 strain was further modified to replace the native hyf promoter region with that from the E. coli hyc operon encoding Hyd-3. This new strain (AF01, Table 1) was then extensively modified, first with the genetic removal of hydrogenase-3 activity (resulting in strains AF02 and AF03, Table 1), then by the additional deletion of the gene encoding the hydrogenase-1 catalytic subunit (yielding strains AF04 and AF06, Table 1). Culturing of all of these strains in triplicate 5 ml Lysogeny Broth (LB) supplemented with 0.8 % (w/v) glucose in sealed Hungate tubes for 16 h at 37 °C demonstrated that replacement of the hyf promoter region with that of hyc did not result in detectible H2 production from Hyd-4 (Table 1).

Table 1.

Rational engineering of the E. coli hyf operon does not induce H2 production

E. coli K-12 strain	Relevant genotype	Source	H2 production
MG1655	F-, λ-, rph-1	[24]	Positive
MG056G1	as MG1655, hyfG His	This Work	Positive
AF01	as MG1655, hyfG His, Phyc::hyfA	This Work	Positive
AF02	as MG1655, hyfG His, Phyc::hyfA, ΔhycA-I:: KanR	This Work	Negative
AF03	as MG1655, hyfG His, Phyc::hyfA, ΔhycA-I	This Work	Negative
AF04	as MG1655, hyfG His, Phyc::hyfA, ΔhycA-I, ΔhyaB::KanR	This Work	Negative
AF06	as MG1655, hyfG His, Phyc::hyfA, ΔhycA-I,ΔhyaB	This Work	Negative
FTE001	as MG1655, hyfG His, Phyc::hyfA, ΔhycA-I, ϕhyfG (nt 1–1569)::hycE (nt 1611–1707)	This Work	Negative
MG059e1	as MG1655, hycE His	[25]	Positive
MGE1dI	as MG1655, hycE His, ΔhycI	This Work	Negative
MC4100	F−, araD139, Δ(argF-lac)169, λ−, e14−, flhD5301, Δ(fruK-yeiR)725(fruA25), relA1, rpsL150(StrR), rbsR22, Δ(fimB-fimE)632(::IS1), deoC1	[27]	Positive
FTD147	as MC4100, ΔhyaB, ΔhybC, ΔhycE	[16]	Negative
AF05	as MC4100, ΔhyaB, ΔhybC, ΔhycE, Phyc::hyfA	This Work	Negative
FTE002	as MC4100, ΔhyaB, ΔhybC, ΔhycE, Phyc::hyfA, ϕhyfG (nt 1–1569)::hycE (nt 1611–1707)	This Work	Negative
FTE003	as MC4100, ΔhyaB, ΔhybC, ΔhycE, PT5::hyfA	This Work	Negative
FTE004	as MC4100, ΔhyaB, ΔhybC, ΔhycE, PT5::hyfA, ϕhyfG (nt 1–1569)::hycE (nt 1611–1707)	This Work	Negative
FTE005	as MC4100, ΔhyaB, ΔhybC, ΔhycE, PT5::hyfA, hyfG His	This Work	Negative
FTE006	as MC4100, ΔhyaB, ΔhybC, ΔhycE, PT5::hyfA, hyfG His, ϕhyfG (nt 1–1569)::hycE (nt 1611–1707)	This Work	Negative
FTE007	as MC4100, ΔhyaB, ΔhybC, ΔhycE, ϕhyfG (nt 1–1569)::hycE (nt 1611–1707)	This Work	Negative

*E. coli strains were grown under anaerobic fermentative conditions in LB medium supplemented with 0.8 % (w/v) d-glucose at 37 °C for 16 h. Production of molecular H2 in the culture headspace was determined by gas chromatography. Hungate tube headspace gas was injected into a 500 µl loop and separated through a 5A molecular packed column before thermal conductivity detection. A hydrogen standard curve was generated using N2:H2 mixes [23].

LB, Lysogeny Broth.

K-12 strain

as MG1655, hyfG His, P::hyfA

as MG1655, hyfG His, P::hyfA, ΔhycA-I:: KanR

as MG1655, hyfG His, P::hyfA, ΔhycA-I

as MG1655, hyfG His, P::hyfA, ΔhycA-I, ΔhyaB::KanR

as MG1655, hyfG His, P::hyfA, ΔhycA-I,ΔhyaB

as MG1655, hyfG His, P::hyfA, ΔhycA-I,

as MC4100, ΔhyaB, ΔhybC, ΔhycE, P::hyfA

as MC4100, ΔhyaB, ΔhybC, ΔhycE, P::hyfA,

Next, an alternative K-12 parental strain (based on MC4100 [26, 27]) was tested. The FTD147 strain (deleted for the genes encoding the catalytic subunits of Hyd-1, -2 and -3 [16]) was modified by replacement of the native hyf promoter with that of the strong T5 promoter from the pQE plasmid series (yielding strain FTE003, Table 1). Growth of this strain under fermentative conditions did not result in any detectible H2 production from Hyd-4 (Table 1).

Finally, it was considered that potential problems with HyfG processing could be leading to synthesis of an immature, inactive Hyd-4. This hypothesis is based on the fact that the hyf operon encodes no specific maturation protease and the reasonable possibility that HycI might not recognize HyfG as a substrate. In order to test this hypothesis, with the aim of forcing HycI to recognize and activate HyfG, a series of strains were carefully constructed where the C-terminal assembly peptide of HycE was added to the mature sequence of HyfG (Table 1, Fig. 1c). Careful genetic engineering generated a ϕhyfG::hycE fusion sequence that would comprise the first 1569 nucleotides of hyfG precisely in-frame with hycE nucleotides 1611–1707 and retaining the ribosome binding site and initiation codon on the downstream hyfH gene to mitigate against potential polar effects. The resulting protein sequence is shown in Fig. 1c. This construct was transferred to the chromosome of a number of promoter-engineered strains (note that these all remain hycI +) using the technique of homologous recombination [22]. No H2 production from Hyd-4 was detected in any of the engineered large subunit fusion strains (Table 1). Taken altogether, the strain-engineering experiments suggest that additional, unknown, biosynthetic problems are hindering assembly of Hyd-4.

Clearly, making progress in the understanding the biochemistry of Hyd-4-like enzymes requires an alternative model system. Recently, group 4A [NiFe]-hydrogenases from [28], Trabulsiella guaensis [29], [30], [31] and [32] have been identified as possible candidates for study of this [NiFe]-hydrogenase group. Our sequence analysis suggests that genetic loci encoding each of these ‘hyf-type’ enzymes contained a hycI-like gene (Fig. 1d). Indeed, bioinformatic analysis of group 4A hydrogenases revealed only four organisms that encode both a Hyd-3 and Hyd-4 orthologue within their respective genomes: Escherichia coli; Shimwellia blattae; Hafnia alvei; and Koskonia radicincitans [11, 33]. In every case only one endopeptidase gene is found within the hyc-like operons, and none could be identified within the hyf-like operons (Fig. 1d, organisms linked by the red line). This suggests that a single HycI-like protease may be sufficient for Hyd-4 biosynthesis, given that a second copy is never conserved.

SCRI1043 is a γ-Proteobacterium that contains an active FHL-2 and Hyd-4 encoded by a hyf operon (Fig. 1a), but no FHL-1 or Hyd-3 isoenzyme [28]. Unlike the E. coli hyf operon, the SCRI1043 hyf operon encodes HyfK (HyfK, Fig. 1a), which shares 74 % overall sequence identity with HycI (HycI). Due to this sequence similarity it was considered that these endopeptidases could be tested for their ability to activate either the Hyd-3-type and the Hyd-4-type hydrogenase. To begin, both hycI and hyfK genes were cloned separately in to pQE80L (AmpR) expression vectors using standard PCR and molecular cloning techniques. Both plasmids, and a vector control, were used to transform the ΔhycI strain MGE1dI (Table 1). MGE1dI is based on MG059e1 (as MG1655, hycE His [25]) but carries an unmarked in-frame deletion in hycI. The transformed strains were grown in triplicate 5 ml LB 0.2 % (w/v) formate cultures, with or without addition of 1 mM IPTG, anaerobically in sealed Hungate tubes, for 24 h at 37 °C, before GC analysis of the culture headspace. The MGE1dI (ΔhycI) strain of , containing empty vector control, displayed no physiological FHL-1 activity and did not evolve H2 gas under fermentative conditions (Fig. 2a). However, incorporation of either hycI or hyfK in the ΔhycI strain rescued H2 production (Fig. 2a). These data demonstrate that the hyfK gene product can facilitate the maturation of the Hyd-3 enzyme.

Fig. 2.

HyfK can activate Hyd-3, and HycI can activate Hyd-4. (a) strains MC4100 (FHL-1+) and MGE1dI (ΔhycI) were transformed separately with plasmids harbouring hycI, hyfK or a vector control (pQE80). Strains were grown anaerobically in LB medium supplemented with 0.2 % (w/v) formate, ampicillin and 1 mM IPTG where indicated (+) for 24 h at 37 °C. (b) strains PH002 (ΔhybC, FHL-2+) and PH006 (ΔhybC, ΔhyfK) were transformed with plasmids containing hycI, hyfK or pQE80. Strains were grown anaerobically in low-salt LB (LSLB) medium supplemented with ampicillin and 1 mM IPTG where indicated (+) for 48 h at 24 °C. (c) strains MGE1dI (ΔhycI), FTE003 (ΔhyaB, ΔhybC, ΔhycE, PT5::hyfA) and FTE004 (ΔhyaB, ΔhybC, ΔhycE, PT5::hyfA, ϕhyfG::hycE) were transformed with plasmids containing hycI, hyfK or a vector control (pQE80). Strains were grown anaerobically in LB medium supplemented with 0.8 % (w/v) glucose, ampicillin and 1 mM IPTG for 16 h at 37 °C. In all cases, H2 headspace samples were extracted and analysed by gas chromatography (Shimadzu GC2014 using a 5A molecular packed column with thermal conductivity detection). Data was normalized by OD600 and culture volume. Error bars represent sd (n=3).

To study the role of maturation proteases in the activation of Hyd-4/FHL-2, SCRI1043, which contains active FHL-2, was studied [28]. First, a genetic approach was taken to assess the role of hyfK in hydrogen production. A double-mutant strain was constructed, using an allele exchange protocol [28], which carried both ΔhybC and ΔhyfK in-frame deletions (PH006, Table 2 and Supplementary Material). The ΔhybC deletion removes all Hyd-2 activity leaving Hyd-4 as the only active hydrogenase in the bacterium [28]. Next, the ΔhybC ΔhyfK double-mutant (PH006), together with the PH002 parent strain (ΔhybC only), were separately transformed with the pQE80 plasmids containing either hycI or hyfK, or the empty vector as a control. The transformed strains were then grown in triplicate 5 ml low salt LB cultures (5 g l−1 NaCl as opposed to the commonly used 10 g l−1), with or without addition of 1 mM IPTG, in sealed Hungate tubes, fermentatively for 48 h at 24 °C, before GC analysis of the headspace gases. The PH002 parent strain (ΔhybC) was able to generate H2 gas under all conditions (Fig. 2b). However, the ΔhybC ΔhyfK double-mutant was incapable of producing any H2 gas in this experiment when carrying an empty vector (Fig. 2b). This shows the hyfK protease gene is essential for FHL-2 and Hyd-4 activity in SCRI1043. Moreover, the ΔhybC ΔhyfK double-mutant strain was clearly rescued for H2 production by inclusion of either hycI or hyfK (Fig. 2b). These data demonstrate that Hyd-4 isoenzymes do require a maturation step for successful biosynthesis and they also suggest that, in the rare cases where an organism has the capability to produce both FHL-1 and FHL-2, that a single copy of hycI should be sufficient for this task.

Table 2.

Mutagenesis of the P. atrosepticum hyf operon

P. atrsosepticum strain	Relevant genotype	Source	H2 production
SCRI1043	wild-type	[48]	Positive
PH002	as SCRI1043, ΔhybC	[28]	Positive
PH006	as SCRI1043, ΔhybC, ΔhyfK	This Work	Negative

*P. atrosepticum strains were grown under anaerobic fermentative conditions in low salt (LS) LB medium supplemented with 0.8 % (w/v) d-glucose at 24 °C for 48 h. Production of molecular H2 in the culture headspace was determined by GC [28]

GC, Gas Chromatography; LB, Lysogeny Broth; LSLB, Low Salt Lysogeny Broth.

This compatibility of HycI and HyfK for activation of either Hyd-3 or Hyd-4 points strongly towards the idea that HycI should be capable of maturation of the endogenous Hyd-4 found in . In one final attempt to observe Hyd-4 activity in , the hycI and hyfK encoding plasmids, and a vector control, were each used to transform the FTE003 and FTE004 strains encoding the HyfG::HycE fusion proteins (Table 1). All strains were grown in triplicate 5 ml LB 0.8 % (w/v) glucose cultures, with addition of 1 mM IPTG, in sealed Hungate tubes, for 16 h at 37 °C, before GC analysis. Hydrogen production was only detected in the control strains (Fig. 2c), demonstrating that cellular levels of a maturation protease is not the sole factor limiting Hyd-4 activity in .

This work presents the first demonstration of cross-species complementation by hydrogenase maturation endopeptidases, highlighting the close evolutionary relationship between group 4 [NiFe]-hydrogenases and demonstrating the critical importance of the HycI-type protease in the biosynthesis of these enzymes. These data are in line with studies showing a endopeptidase for a group 1D hydrogenase was able to activate a different group 1D enzyme within the same organism () [34], and one endopeptidase was able to activate both a group 3B hydrogenase and group 4D hydrogenase within the same organism () [35].

Though the proteolytic maturation schedule for [NiFe]-hydrogenases is now dogma, there are known and emerging variations on the canonical pathway for large subunit biosynthesis. Protelolytic processing is not required for all [NiFe]-hydrogenases, such as examples of the H2-sensing, Ech- and CODH-linked hydrogenases [36-39]. Indeed, recent genetic engineering work showed that removal of the C-terminal assembly peptide from the membrane bound hydrogenase (MBH) in () did not disrupt cofactor insertion and resulted in no loss of hydrogenase-specific activity [40]. Given that in a maturation protease was found to retain the ability to recognize and bind to a large subunit completely lacking the maturation peptide [34], perhaps it should be considered that the maturation protease has a role in hydrogenase biosynthesis beyond the simple cleavage of the C-terminal extension. This could certainly be tested in the system [40] by deleting the gene encoding the processing protease (HoxM [41]) in the stain already lacking the hydrogenase assembly peptide and observing any changes to hydrogenase activity.

It is becoming increasingly clear that the C-terminal assembly peptide may not be the key recognition motif for the protease [34, 42]. Early work showed that swapping of the HycE (Hyd-3) assembly peptide for that of HybC (Hyd-2) led to a ‘dead-end’ fusion protein that could not be processed by any maturation protease tested [43]. While more recently, swapping the HybC (Hyd-2) assembly peptide for that of HyaB (Hyd-1) did not lead to any changes in the requirement for the Hyd-2-specific protease (HybC) for maturation [42, 44].

In conclusion, this work has demonstrated that group 4 [NiFe]-hydrogenases require a functional hycI-like accessory gene for correct biosynthesis. A model bacterium ( SCRI1043), which contains an active Hyd-4 and FHL-2 as the only formate hydrogenlyase activity, required the presence of the native hyfK gene product for maturation. The E. coli hycI gene could substitute for P. atrosepticum hyfK if supplied on a plasmid, providing an explanation of why it is that in rare examples of organisms that contain both an FHL-1 and an FHL-2 only one copy of a hycI-like gene is conserved.

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