Development and Retention of a Primordial Moonlighting Pathway of Protein Modification in the Absence of Selection Presents a Pu

Home , GCSH, Glycine cleavage system, Protein moonlighting

Development and retention of a primordial INAUGURAL ARTICLE moonlighting pathway of protein modification in the absence of selection presents a puzzle

Xinyun Caoa,1, Yaoqin Hongb,1, Lei Zhub,2, Yuanyuan Hua, and John E. Cronana,b,3

aDepartment of Biochemistry, University of Illinois at Urbana–Champagne, Urbana, IL 61801; and bDepartment of Microbiology, University of Illinois at Urbana–Champagne, Urbana, IL 61801

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2017.

Contributed by John E. Cronan, December 7, 2017 (sent for review October 25, 2017; reviewed by Sean T. Prigge and Eric Smith) Lipoic acid is synthesized by a remarkably atypical pathway in which sulfur protein, LipA. LipA donates two sulfur atoms to carbons 6 and the cofactor is assembled on its cognate proteins. An octanoyl 8 of octanoate to give a dihydrolipoyl moiety which becomes oxidized moiety diverted from fatty acid synthesis is covalently attached to to lipoyl-LD (Fig. 1A). Hence, lipoic acid is an atypical enzyme co- the acceptor protein, and sulfur insertion at carbons 6 and 8 of the factor in that it is assembled on its cognate proteins rather than being octanoyl moiety form the lipoyl cofactor. Covalent attachment of this wholly assembled before its covalent attachment, as is the case with cofactor is required for function of several central metabolism biotin, another covalently attached coenzyme (5). enzymes, including the glycine cleavage H protein (GcvH). In Bacillus The straightforward two-enzyme, LipB plus LipA, E. coli lip- subtilis, GcvH is the sole substrate for lipoate assembly. Hence lipoic oate synthesis pathway is found in many, but not all, bacterial acid-requiring 2-oxoacid dehydrogenase (OADH) proteins acquire the species. A more complex pathway is found in Bacillus subtilis (6– cofactor only by transfer from lipoylated GcvH. Lipoyl transfer has been 9). The impetus to study this bacterium was the lack of a gene Escher- argued to be the primordial pathway of OADH lipoylation. The encoding a LipB homolog (6), although a validated LipA homolog ichia coli pathway where lipoate is directly assembled on both its GcvH

was present (9). Moreover, the B. subtilis genome encoded what BIOCHEMISTRY and OADH proteins, is proposed to have arisen later. Because roughly appeared to be three lipoate ligase homologs, the paradigm of 3 billion years separate the divergence of these bacteria, it is surprising which, E. coli LplA, scavenges lipoate from the environment (8). that E. coli GcvH functionally substitutes for the B. subtilis protein in Screening of a plasmid library of B. subtilis genome fragments for lipoyl transfer. Known and putative GcvHs from other bacteria and eukaryotes also substitute for B. subtilis GcvH in OADH modification. complementation of an E. coli strain lacking LipB gave one of the Because glycine cleavage is the primary GcvH role in ancestral bacteria putative lipoate ligase genes. However, the gene encoded an that lack OADH enzymes, lipoyl transfer is a “moonlighting” function: octanoyl transferase called LipM that had no ligase activity and that is, development of a new function while retaining the original virtually no sequence resemblance to E. coli LipB (6). Another function. This moonlighting has been conserved in the absence of putative lipoate ligase gene (LplJ) encoded a true lipoate ligase, selection by some, but not all, GcvH proteins. Moreover, Aquifex aeolicus encodes five putative GcvHs, two of which have the moonlighting Significance function, whereas others function only in glycine cleavage. The LipB–LipA and lipoyl-relay pathways seem restricted to lipoic acid | glycine cleavage | enzyme moonlighting | aerobic gram-negative and gram-positive bacteria, respectively. metabolism | C1 metabolism Braakman and Smith have persuasively argued that the lipoyl- relay pathway involving transfer of lipoic acid from lipoylated ipoic acid is an essential sulfur-containing coenzyme required GcvH is the primordial pathway and that the LipB–LipA path- Lby key enzymes of central metabolism in all three domains of way arose later. The chemically challenging nature of lipoyl life (1, 2). Unlike most coenzymes, lipoic acid functions only after transfer strengthens this argument. Given the roughly 3 billion covalent attachment to its cognate enzyme proteins. These are the years since the two groups of bacteria diverged, it is un- E2 subunits of the 2-oxoacid dehydrogenase (OADH) complexes expected and puzzling that the lipoyl transfer properties of and the H proteins (called GcvH or GcsH, depending on the or- many GcvHs—those from eukaryotes and bacteria, including ganism) of the glycine cleavage system (glycine decarboxylase in Escherichia coli, and the deep-branching hyperthermophilic Aquifex aeolicus— plants), all of which have been well studied (1). Although the OADH chemoautotrophic bacterium, have lipoyl- Bacillus subtilis lipoyl domains (LDs) and the GcvH proteins both contain a strictly relay activity in . This provides a remarkable “ ” conserved lysine residue to which lipoic acid is attached and have case in which a protein moonlighting function is found remarkably similar structures, the proteins have only low sequence throughout evolution without selection for the function. identities (1, 3). Lipoic acid attachment to these proteins is via an Author contributions: X.C., Y. Hong, and J.E.C. designed research; X.C., Y. Hong, L.Z., and amide linkage formed between the lipoic acid carboxyl group and Y. Hu performed research; L.Z. and Y. Hu contributed new reagents/analytic tools; Y. Hong the conserved lysine e-amino group (1, 3). Once attached, the lipoyl and J.E.C. analyzed data; and X.C. and J.E.C. wrote the paper. moiety becomes competent for enzyme catalysis. The terminal Reviewers: S.T.P., The Johns Hopkins Bloomberg School of Public Health; and E.S., Santa dithiolane ring becomes reduced and acylated with a reaction in- Fe Institute. termediate and the lipoyl-lysine arm serves to shuttle reaction inter- The authors declare no conflict of interest. mediates between the active sites of the multienzyme complexes (3). Published under the PNAS license. Lipoic acid assembly is best understood in Escherichia coli,where 1X.C. and Y. Hong contributed equally to this work. only two enzymes are required. LipB, an octanoyl transferase trans- 2Present address: College of Life Sciences, Shandong Agricultural University, Taian, fers the octanoyl moiety from fatty acid biosynthetic intermediate 271018 Shandong, China. octanoyl-acyl carrier protein (ACP), to the specific lysine side chain of 3To whom correspondence should be addressed. Email: [email protected]. GcvH or an OADH LD (1, 4). The resulting octanoyl-protein is a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. substrate for the sulfur insertion step catalyzed by the radical iron- 1073/pnas.1718653115/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1718653115 PNAS Early Edition | 1of9 Downloaded by guest on September 30, 2021 Fig. 1. Lipoic acid synthesis and scavenging in E. coli and B. subtilis.(A) The E. coli lipoic acid synthetic pathway requires only two proteins, LipB and LipA. The LipB octanoyl-ACP transferase transfers the octanoyl moiety from the fatty acid biosynthetic intermediate, octanoyl-ACP, to both OADH LDs and GcvH, where LipA-catalyzed sulfur insertion converts the octanoyl moiety to lipoate. (B) The B. subtilis lipoic acid synthetic pathway requires four proteins (three enzymes plus the GcvH protein, on which lipoate is assembled and donated to the OADH proteins) rather than the two enzymes of the simpler E. coli pathway (A). Like E. coli, B. subtilis has the LipA sulfur insertion enzyme and an octanoyl transferase, LipM, although LipM is an isozyme rather than a homolog of the E. coli LipB octanoyl transferase. The octanoylated GcvH then becomes a substrate for LipA-catalyzed sulfur insertion at carbons 6 and 8, as in A. The lipoyl moiety is then relayed from GcvH to the OADH subunits by an amidotransferase called LipL. (C) Both bacteria scavenge lipoate from the medium by use of the reaction shown for the E. coli lipoate ligase, LplA. The B. subtilis ligase is LplJ (8). The ligases (which are 33% identical) are required for lipoate supplementation of strains blocked in lipoic acid synthesis. E. coli LplA efficiently lipoylates both its cognate GcvH and the OADH domains (43), whereas in B. subtilis GcvH is a much better substrate for LplJ than are the OADH proteins (8).

whereas the third putative lipoate ligase gene (lipL) remained an LDs. Indeed when LipL, the third putative B. subtilis lipoate li- enigma, although it was essential for lipoate synthesis (8). The gase, was purified, the protein lacked ligase activity but was found enzymological characterization of LipM presented a quandary in to transfer an octanoyl moiety from its amide linkage on GcvH to that the purified enzyme was unable to transfer octanoate from the PDH LD using an acyl-enzyme (acyl-cysteine) intermediate octanoyl-ACP to its cognate pyruvate dehydrogenase (PDH) LDs, (7). These biochemical experiments argued that GcvH was an although the proteins were good substrates for the E. coli LipB obligate intermediate in lipoylation of the B. subtilis OADH octanoyl transferase (7). A concern was the possibility that LipM proteins, and therefore B. subtilis ΔgcvH strains should be unable recognition of the B. subtilis PDH domains required assembly of to make lipoate and be auxotrophic for lipoate. This was the case: the E2 subunit into the very large PDH complexes that contain the small GcvH protein is required for both lipoylation of the many copies of E2, plus the E1 and E3 subunits. To avoid the B. subtilis subunits (PDH for aerobic metabolism and the branched- possibility of a complex being the substrate, GcvH, a protein of chain OADH for fatty acid synthesis) and for glycine cleavage (7). only 127 residues (and thus only slightly larger than the OADH Hence, a small protein of single-carbon metabolism has a second LDs of ∼80 residues), was tested as a LipM substrate. In contrast “moonlighting” (10–12) function that, for simplicity, we will refer to the cognate PDH LDs, GcvH turned out to be an excellent to as the lipoyl-relay pathway. Therefore, although it is a protein LipM substrate (7), which raised the possibility that GcvH might of only 127 residues, the B. subtilis glycine cleavage H protein somehow act as an intermediate between LipM and the OADH (GcvH) has three separate functions in central metabolism. Note

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1718653115 Cao et al. Downloaded by guest on September 30, 2021 that essentially the same pathway has recently been demonstrated The five putative A. aeolicus GcvH proteins were problematic

in Staphylococcus aureus (13), and phenotypic analyses of Sac- both in their number and lack of genome context. The lack of INAUGURAL ARTICLE charomyces cerevisiae argue that this yeast also may use a similar genome context is characteristic of the A. aeolicus genome (21). pathway (14). For example, although the genes encoding the glycine cleavage Brocklehurst and Perham (15) first deduced that despite low system GcvP and GcvT proteins are almost always contiguous sequence identities (15–20%), the OADH LD structures could genes in bacteria, this is not the case in A. aeolicus, where even the be used to accurately predict the pea GcvH structure. Although a two GcvP subunits are encoded remotely. The only clue as to similar low sequence identity is seen between the E. coli OADH which of the five putative GcvHs might be the “real” GcvH is LDs and its GcvH, the LipB octanoyl transferase efficiently GcvH4, which is encoded next to the GcvP2 subunit gene, and modifies both proteins (16, 17). This is not the case with the indeed this protein gave robust complementation. The GcvH1 B. subtilis LipM octanoyl transferase, which efficiently modifies protein also had complementation activity but more weakly than its cognate GcvH but fails to modify its cognate OADH LDs (7), GcvH4. The GcvH2, GcvH3, and GcvH5 proteins were completely and thus the OADH domains must obtain bound lipoate from inactive in complementation, as were all three of the OADH LDs GcvH by LipL-catalyzed transfer (7) (Fig. 1B). This lipoyl tested (those from E. coli, S. coelicolor,andH. sapiens). transfer reaction is chemically challenging because the LipL To exclude the possibility that the lack of complementation active site thiol must attack a kinetically stable amide bond. was due to poor protein expression, we constructed a set of Given this challenge, it seems likely that GcvH provides an en- B. subtilis ΔgcvH derivatives in which the proteins that had failed vironment that facilitates the LipL reaction and that the OADH to complement (GcvH2, GcvH3, and GcvH5 from A. aeolicus)and domains lack this property. To test this hypothesis, we replaced the OADH LDs from E. coli, S. coelicolor, and H. sapiens were the B. subtilis gcvH with genes encoding diverse GcvH and tagged with a C-terminal hexahistidine sequence to allow im- OADH LD proteins and tested for restoration of growth in the munological detection. The B. subtilis ΔgcvH strain expressing a absence of lipoic acid. We also tested the proteins as acceptors of tagged E. coli GcvH provided a positive control. As shown in Fig. 3B, LipM-catalyzed octanoyl transfer in vitro. Substitution of E. coli the three GcvH proteins (GcvH2, GcvH3, and GcvH5) from GcvH for the B. subtilis protein would be particularly interesting A. aeolicus and three LDs of E. coli, S. coelicolor, and H. sapiens because the E. coli does not use the lipoyl-relay pathway (Fig. that failed to complement the B. subtilis ΔgcvH strain were all 1A). Successful substitution would indicate that E. coli GcvH detected by Western blotting and thus were expressed. Hence, retains the ability to support lipoyl relay despite a lack of se- the failure to complement could not be attributed to a lack lection for this function. Moreover we also tested the seemingly BIOCHEMISTRY of expression. redundant five copies of GcvH encoded in the small genome (1.5 Mb) of the deep-branching bacterium, Aquifex aeolicus. The Proteins That Failed to Complement Cannot be Modified by LipM. These proteins were analyzed because Braakman and Smith (18) The inability of a given GcvH or OADH LD to complement the developed their hypothesis that GcvH is the primordial lip- lipoate requirement of the B. subtilis ΔgcvH strain could be due to oylated protein based on their analysis of A. aeolicus. To our an inability to accept octanoate in the reaction catalyzed by LipM surprise, two of these proteins were able to provide lipoyl-relay or to transfer lipoate from GcvH to the OADH subunits, the LipL function to B. subtilis. reaction (Fig. 1). It is possible that a given protein could be inactive Results in both enzymatic reactions, but in the absence of LipM activity, LipL would have no acceptor substrate for transfer. Hence, the Complementation of B. subtilis ΔgcvH Lipoate Auxotrophy by Genes ability of a given protein to be modified by LipM but unable to Encoding Alternative GcvH and OADH LD Proteins. To test the lipoyl- complement OADH lipoylation could be attributed to defective relay abilities of known and putative GcvH proteins from diverse LipL-catalyzed transfer. On the other hand, modification by LipM organisms, we used a B. subtilis ΔgcvH strain (NM20) in which would be expected for the proteins that showed complementation. deletion of the gcvH gene resulted in a requirement for lipoic acid (7–9). To test GcvH and LD proteins from other organisms To test these predictions, we purified the GcvH proteins of E. coli, for the ability to substitute for B. subtilis GcvH, the single GcvHs P. sativum, H. sapiens, S. coelicolor, the two complementing and of E. coli, Streptomyces coelicolor, Pisum sativum (pea), Homo three inactive putative GcvHs from A. aeolicus, plus the LD of sapiens, all five putative GcvHs from A. aeolicus (Fig. 2), together H. sapiens PDH by nickel affinity chromatography. Some purified with LD proteins of E. coli, S. coelicolor,andH. sapiens,were proteins migrated as doublets when analyzed on SDS/PAGE gel tested. Each gene was cloned and integrated into the chromosomal (Fig. 4A), which is attributed to deamidation during purification amyE site of the B. subtilis ΔgcvH strain under the isopropyl-β-D- and did not affect modification (22, 23). Note that GcvH and LD thiogalactopyranoside (IPTG)-dependent promoter, Pspac. proteins migrate on SDS/PAGE at rates expected for proteins of This set of B. subtilis ΔgcvH strains was tested for relief of about twice their size. This is because both GcvH and LD are very ∼ lipoic acid auxotrophy by colony formation in the absence of acidic proteins (calculated pIs 3.9), and hence bind abnormally low amounts of SDS relative to the protein standards. We tested lipoic acid on minimal medium plates containing glucose as 14 carbon source (Fig. 3A). Slight background growth was observed each protein for its ability to accept LipM-catalyzed [1- C] on plates lacking IPTG, which is readily attributed to the known octanoyl-transfer in vitro. The products were then analyzed by basal expression of the Pspac promoter and the low levels of electrophoresis using conformationally sensitive urea PAGE (20%) lipoic acid required for growth. All of the strains grew well in the rather than SDS/PAGE because some GcvH proteins comigrated presence of lipoic acid, indicating that the expressed proteins with B. subtilis acyl-ACP in the SDS system. Upon autoradio- were not toxic to growth (Fig. 3A). Interestingly, the GcvH graphic analysis of acceptor protein modification the GcvH proteins of E. coli and S. coelicolor supported robust growth of proteins of B. subtilis, E. coli, S. coelicolor, H. sapiens, P. sativum, the B. subtilis ΔgcvH strain in the absence of lipoic acid, despite as well as GcvH1 and GcvH4 from A. aeolicus, were readily the fact that in their native hosts these GcvHs do not participate octanoylated by LipM (Fig. 4B). In contrast, no octanoyl transfer in lipoate synthesis. Expression of two eukaryotic GcvH proteins, to the A. aeolicus GcvH2, GcvH3, and GcvH5 proteins or to the the human (H. sapiens) and pea (P. sativum) proteins, both of LD of H. sapiens PDH was observed. These data indicate that the which are known to function in glycine cleavage (19, 20), also proteins that are unable to restore lipoic acid synthesis in vivo fail supported robust growth in the absence of lipoic acid. Indeed, to complement due to the lack of modification by LipM. Note the pea GcvH gave strong growth even in the absence of that the [1-14C]octanoyl-ACP was generated in situ using the IPTG induction. AasS acyl-ACP synthetase, as described in Materials and Methods.

Cao et al. PNAS Early Edition | 3of9 Downloaded by guest on September 30, 2021 Fig. 2. Amino acid sequence alignments of known and putative GcvH proteins and the central role of GcvH in the glycine cleavage mechanism. (A) Sequence alignment of GcvH proteins of E. coli, H. sapiens, P. sativum, S. coelicolor, B. subtilis, and five putative A. aeolicus GcvH proteins (GcvH1, GcvH2, GcvH3, GcvH4, GcvH5). Conserved residues are shown as white letters on a red background, and similar residues are shown as red letters in blue boxes. The E. coli GcvH secondary structure (PDB ID code 3A7L) is shown at the top of the panel. The vertical arrow denotes the lysine residue that becomes modified. (B) The glycine cleavge system and the central role of GcvH. The ammonia that provides an assay for GcvH activity is colored blue. Note the L protein that oxidizes the lipoyl cofactor is the same protein as the dihydrolipoyl dehydrogenase (Lpd) of the OADH complexes.

B. subtilis GcvH, the natural substrate of LipM, served as positive source (the NM20 strain requires tryptophan and phenylalanine control in these reactions (6). and grows best when the medium contains a trace amount of acid hydrolyzed casein). The presence of other nitrogen sources gave The Putative GcvH2, GcvH3, and GcvH5 Proteins of A. aeolicus Cannot some background growth of the parental strain that ceased when Replace B. subtilis GcvH in Glycine Cleavage. Glycine cleavage pro- the hydrolyzed casein nitrogen (mainly glutamate) was exhausted, duces 5,10-methylene tetrahydrofolate, CO2,andNH3 (Fig. 2B). whereas in the presence of glycine growth continued robustly. In Bacteria lacking function of one of the gcv genes have two phe- contrast, the B. subtilis ΔgcvH strain NM20 showed only back- notypes. These are an inability to use glycine as a nitrogen source ground growth. Strains expressing each of the GcvH proteins and an inability to supplement serine auxotrophs with glycine in known to function in glycine cleavage (the human, pea, E. coli,and place of serine (the 5,10-methylene tetrahydrofolate produced by B. subtilis proteins) allowed growth of the B. subtilis ΔgcvH strain cleavage of a glycine molecule is used to convert another glycine on glycine as nitrogen source (Table 1). The strain expressing molecule to serine). We have taken advantage of the first of these A. aeolicus GcvH4grewwellwithglycineasanitrogensource(Fig. phenotypes to assess the abilities of the various GcvH and OADH 5), consistent with the genomic location of the encoding gene. LD proteins to function in B. subtilis glycine cleavage. The am- Therefore, each of these proteins had both glycine cleavage and monium sulfate of Spizizen salts minimal medium was replaced lipoyl-relay activity. The other annotated A. aeolicus GcvH proteins with potassium sulfate and glycine was added as the major nitrogen gave a variety of glycine cleavage phenotypes (Fig. 5 and Table 1).

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1718653115 Cao et al. Downloaded by guest on September 30, 2021 Discussion

Despite the well-characterized role of lipoic acid as a protein- INAUGURAL ARTICLE bound coenzyme, the detailed mechanisms of its synthesis and attachment to cognate proteins are still unclear in many organisms, including bacteria and eukaryotes (1). The most straightforward lipoic acid synthesis pathway is the LipB–LipA pathway of E. coli (Fig. 1) and numerous other bacteria, where lipoyl- GcvH functions only in the glycine cleavage cycle. In marked contrast, B. subtilis GcvH is not only a glycine cleavage system component, but also has a moonlighting function that is required for lipoylation of the OADH enzymes required for aerobic metabolism and branched-chain fatty acid synthesis. In their modeling studies of early evolution of metabolism based on the deep-branching hyperthermophilic chemoautotrophic bacterium, A. aeolicus, Braakman and Smith (18) argued that the glycine cleavage system first arose as part of the ancestral glycine/serine pathway and the sole function of lipoic acid was in shuttling the intermediates of this pathway. These authors further argued that utilization of lipoate in glycine cleavage preceded its use by the OADH enzymes because the TCA cycle is not essential in many bacteria, whereas glycine cleavage is almost totally conserved. Their case was further buttressed by noting that those bacteria that lack the oxidative TCA cycle should lack LipL because there would be no OADH proteins to modify, and this is the case (18). Moreover, the few bacteria that lack the genes of the glycine cleavage system would be expected to also lack the genes of lipoic acid synthesis and scavenging, and again this is the case. Taken together, these considerations argue BIOCHEMISTRY strongly that the B. subtilis pathway is derived from the ancestral pathway of lipoic acid attachment (18). It follows that transfer of lipoic acid via the lipoyl-relay pathway to the OADH proteins can be considered as an energetically unfavorable “make-do” arrangement that was superseded by the energetically favorable direct transfer of octanoate to the OADH (and GcvH) proteins by broad specificity octanoyl transferases, such as E. coli LipB. In Fig. 3. (A) Growth of B. subtilis ΔgcvH strains expressing genes encoding B. subtilis, transfer of lipoic acid from GcvH to the OADH various GcvH and LD proteins. The genes were integrated into the chro- proteins requires LipL, which based on sequence and structural mosomal amyE site of the B. subtilis ΔgcvH strain under control of Pspac similarities, seems to have been derived from LipM, presumably promoter. The B. subtilis ΔgcvH NM20 strain is auxotrophic for lipoic acid and by duplication of LipM with one of the copies subsequently ac- thus growth in the absence of lipoic acid-denoted complementation. Colony quiring amidotransferase activity (10, 11, 18, 24, 25). Indeed, the formation was scored after 36 h at 37 °C on minimal agar plates with glucose as the sole carbon source in the presence or absence of lipoic acid. The IPTG above lipoate metabolism enzymes plus the biotin protein ligases concentration was 0.5 mM. The wild-type (JH642) and B. subtilis ΔgcvH that constitute Pfam PF03099 are all constructed on the same (NM20) strains served as positive and negative controls, respectively. scaffold, albeit from diverse sequences and extra substrate- (B) Western blot analysis of GcvH and LD expression. The strains expressing activating domains in the ligases (1). As mentioned above, proteins that failed to complement growth of the B. subtilis ΔgcvH strain LipB and LipM share almost no sequence conservation, and were grown for 22 h in minimal medium with lipoic acid and 0.5 mM IPTG. moreover the enzymes diverge in that their active site cysteine Crude cell extracts were separated on SDS/PAGE gels and immunoblotted residues are on different loops of the common scaffold (6). with antihexahistidine antibody (Materials and Methods). Crude extracts of Hence, alteration of a lipM gene to encode LipL activity may the B. subtilis ΔgcvH amyE:: Pspac-E. coli gcvH-His6 strain (XC173) were used as the positive control. Crude extracts of B. subtilis ΔgcvH derivatives have been easier than conversion of either LipM or LplJ into a expressing the various gcvH and OADH LD proteins are as indicated. protein having LipB activity. At present LipB homologs are found only in the proteobacteria, whereas LipM and LipL homologs are found only in the Firmi- The strain expressing the GcvH2 protein failed to grow on the cutes. Based on sequence analysis of 32 proteins believed essential glycine medium and the strain expressing GcvH5 gave only barely for growth (and hence resistant to horizontal gene transfer), detectable growth, and thus these proteins have either no or ex- Battistuzzi et al. (26) argue that these bacterial phyla diverged tremely weak glycine cleavage activity and lack lipoyl-relay activity. about 0.7 billion y before the rise in oxygen levels (2.3 billion y The strain expressing the GcvH1 protein failed to grow on the ago). Emergence of the organelles of bacterial origin, mitochon- glycine medium, although it has weak lipoyl-relay activity. In con- dria, and chloroplasts, which contain the lipoate-modified pro- trast, the strain expressing the GcvH3 protein, which lacks lipoyl- teins, is thought to have occurred at least 1.5 billion y ago (27). relay activity, grew on the glycine medium (Fig. 5). These data Hence, there was ample time available for genetic drift in the argue that the residues responsible for lipoyl relay and glycine sequences of the bacterial and eukaryotic GcvHs we tested. In- cleavage are not identical. Although most GcvH proteins have deed, genetic drift is consistent with the low levels of strictly both functions, some have only lipoyl-relay activity or glycine conserved residues (<10% or less, if the glycine residues required cleavage activity. To our knowledge this evidence that foreign for the turns of antiparallel β-sheets are put aside). However, GcvH proteins can function with noncognate GcvP and GcvT despite sequence drift the ability of GcvH to function in lipoate proteins is unique. As expected, the OADH LD proteins were relay clearly arose early in bacterial evolution because two of the inactive in glycine cleavage. A. aeolicus proteins (GcvH4 and GcvH1) functionally replace

Cao et al. PNAS Early Edition | 5of9 Downloaded by guest on September 30, 2021 A B GcvH GcvH4 GcvH5 GcvH2 GcvH3 GcvH GcvH1 LD GcsH GcvH GcvH gcvH aeolicus aeolicus aeolicus kDa coelicolor A. A. No A. aeolicus E. coli E. coli B. sublis H. Sapiens A. A. A. aeolicus P. savum A. A. H. sapiens S.

250 150 100 75 50

37 25 20 15

1 2 3 4 5 6 L 7 8 9 10 11 [1-14C]octanoyl-ACP

Fig. 4. Purification and octanoyl transferase assays of GcvH and LD proteins. (A) Purification of the GcvH and LD proteins. The proteins were purified as described in Materials and Methods and analyzed by SDS/PAGE on a 4–20% polyacrylamide gel. The molecular weights of the prestained broad-range protein standards (Bio-Rad) are indicated. 1: B. subtilis GcvH, 2: H. sapiens LD, 3: E. coli GcvH, 4: S. coelicolor GcvH, 5: P. sativum GcvH, 6: H. sapiens GcsH, 7–11: GcvH1, GcvH2, GcvH3, GcvH4, and GcvH5 from A. aeolicus, respectively. (B) Assay of [1-14C]octanoyl transfer from [1-14C]octanlyl-ACP to GcvH and LD proteins as indicated in the picture. The [1-14C]octanoyl-ACP was synthesized in situ from [1-14C]octanoate by AasS added to the reaction (ATP was added for the AasS reaction). Synthesis of [1-14C]octanoyl-ACP required [1-14C]octanoate, AasS, ATP and holo-ACP, whereas formation of [1-14C]octanoyl-GcvH/LD required apo- GcvH or LD, LipM and [1-14C]octanoyl-ACP.

B. subtilis GcvH, although this bacterium lacks any OADH sub- be approached by genetic analyses. We seek mutant GcvH pro- strates to accept relayed lipoyl moieties. Moreover, lipoate-relay teins that have lost lipoyl-relay ability while retaining glycine ability has been retained in the GcvH proteins of bacteria (E. coli, cleavage activity and vice versa. S. coelicolor) that have no use for this proficiency because they The putative GcvH protein, GcvH2, which failed to function in utilize a direct and more efficient lipoylation pathway. either lipoyl relay or glycine cleavage must be involved in an- The most straightforward explanation of the curious state of other pathways or is a (temporary?) evolutionary remnant. This affairs in which extremely diverse bacteria (A. aeolicus and may also be true of GcvH5, which has very weak glycine cleavage E. coli) possess an activity that is metabolically extraneous would activity and no lipoyl-relay activity. These two proteins seem be that lipoate-relay ability is “hard-wired’ into GcvH proteins. In closely related to one other and to GcvH3, which has only glycine this scenario, sequences required for glycine cleavage would also cleavage activity (Fig. 6). The GcvH1 protein, which has only play important roles in the lipoyl-relay pathway. However, the lipoyl-relay activity, seems off by itself, whereas the GcvH4 A. aeolicus proteins show this is not the case (Table 1). The two protein, which has both activities, clusters with known GcvH proteins that are active in lipoate relay, GcvH4 and GcvH1, proteins. However, a caveat to these comparisons is that these differ in their glycine cleavage activities. GcvH4 has good ac- are small proteins and thus only a few residue substitutions are tivity, whereas GcvH1 lacks activity. Moreover GcvH3 and needed to alter groupings. As expected from prior analyses (6), GcvH5 have modest and very weak glycine cleavage activities, the OADH LDs and the GcvH proteins (perhaps excepting respectively, but lack lipoate-relay activity. Finally GcvH2 lacks GcvH1) fall into separate clades. both activities. It should be noted that full transcription of bacterial glycine It has been argued that during evolution, acquisition of spe- cleavage system genes proceeds only when high concentrations of cific new interactions is neither difficult nor rare (12). In this glycine are present in the environment. For example, addition of scenario proteins continually gain and lose new activities, only a glycine to a wild-type E. coli culture growing in minimal medium few of which contribute to fitness and are retained. Indeed, ex- results in a 30- to 60-fold increase in glycine cleavage activity (28, amples are known in which one or two residue changes can alter 29). In E. coli the proteins required for glycine cleavage, including enzyme specificity and protein–protein interactions (12). This GcvH, are all encoded in a coordinately regulated operon except view may provide an explanation for the diverse activities of the the dihydrolipoyl dehydrogenase subunit, which is shared with the five annotated A. aeolicus GcvH proteins. It may be that the OADH complexes (28). In B. subtilis, induction of the glycine GcvH structural scaffold permits lipoate-relay ability to arise cleavage pathway is mediated by a glycine riboswitch, which allows frequently and that we have fortuitously “caught” the GcvH1 and transcription in the presence of glycine (30). A problem would GcvH4 proteins before lipoate relay will be lost by the stochastic arise if GcvH were under control of the riboswitch because processes of mutation and drift. Note that the presence of other OADH lipoylation (hence aerobic growth and fatty acid synthesis) A. aeolicus proteins having glycine cleavage activity argues that would occur only in the presence of high glycine concentrations. the proteins capable of lipoate relay would not be required for glycine cleavage. Indeed the presence of five closely similar proteins in A. aeolicus can be viewed as license to allow evolu- Table 1. Functional activities of the putative A. aeolicus GcvH tionary tinkering without loss of a fitness requirement. To extend proteins these considerations to single copy GcvH proteins (e.g., that of Function GcvH1 GcvH2 GcvH3 GcvH4 GcvH5 E. coli) that have lipoyl-relay activity but perform OADH lipoylation by a direct mechanism raises the possibility that the Lipoyl relay + −−+ − residues required for lipoyl relay may aid glycine cleavage and Glycine cleavage −−(+) + ((+)) provide a modest contribution to fitness. Sequence alignments Symbols: The plus signs mean strong activity whereas the minus signs give no obvious clues to explain the diversity among the lipoyl mean no detectable activity. The plus signs in parentheses indicate modest relay and glycine cleavage phenotypes. This puzzle will have to activity (+) or barely detectable activity ((+)).

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1718653115 Cao et al. Downloaded by guest on September 30, 2021 INAUGURAL ARTICLE

Fig. 5. Glycine cleavage activitites of the A. aeolicus GcvH homologs. The medium was a modified Spizizen salts that lacked ammonium sulfate and was supplemented with glucose, 0.01% each of L-phenylalanine and L-tryptophan and 0.015% acid hydrolyzed casein. Either glycine or L-glutamic acid (a pre- ferred nitrogen source for this bacterium) was added at 0.1%. The plates were incubated at 37 °C for 72 h. No discrete colonies were formed by the strains that expressed GcvH1 and GcvH2 (the imperfections are scratches from the streaking), whereas barely visible colonies were formed within the scratches by the strain that expressed GcvH5. Note that this figure is a collage of scans.

However, B. subtilis avoids this predicament because the ribos- Phylogenetic Analysis. The amino acid sequences of different species were witch controls only the genes that encode the GcvP and GcvT retrieved from the National Center for Biotechnology Information. Both the enzymes (30). GcvH is encoded at a distant site in the genome and number of best-scoring sequences and the number of best alignments were set to 1,000. The e-values of the sequences selected were between 10−40 and appears to be constitutively expressed (as required for OADH − 10 50. The phylogenetic tree was constructed using the maximum-likelihood lipoylation). Indeed, a survey of genome sequences indicates that method with the PhyML program (34, 35). PHYLIP Interleaved was used for the genomic location of gcvH in relation to gcvP and gcvT genes alignment. The Bootstrap analysis was set to 1,000 replicates. mayprovideadiagnosticforthemodeofOADHlipoylation.That BIOCHEMISTRY is, when the gene encoding GcvH is remote from those encoding Plasmid and Strain Constructions. The primers used for PCR amplification are the P and T subunits, the bacterium uses the lipoyl-relay pathway, given in Table S2. Strain NM20 was complemented with ectopic gcvHsor whereas in genomes where GcvH is encoded together with the P OADH domains under control of an IPTG-dependent promoter (Pspac). and T subunits, the direct (LipB) pathway is used. Plasmids pXC.045-pXC.056 was constructed by inserting PCR-amplified gcvH Note that a natural lipoate auxotroph, Listeria monocytogenes, or OADH domain gene fragments into pDR111, a gift of D. Rudner, Harvard uses a modified lipoyl-relay pathway. This bacterium acquires Medical School, Boston, MA (36). The five putative gcvH genes from A. lipoic acid from its mammalian host cells and uses a ligase to aeolicus gcvH1 [Kyoto Encyclopedia of Genes and Genomes (KEGG) entry aq_1052; gcvH2, KEGG entry aq_1657; gcvH3, KEGG entry aq_944, gcvH4: attach it to GcvH followed by LipL-catalyzed lipoyl relay to its KEGG entry aq_1108; gcvH5, KEGG entry aq_402], the LD of S. coelicolor OADH proteins (31). Finally, differential lipoylation of GcvH branched-chain 2-oxoacid dehydrogenase E2 (KEGG entry SCO3815), E. coli and the OADH proteins has recently been reported in Plasmo- gcvH (KEGG entry b2904), and S. coelicolor gcvH (KEGG entry SCO5471) were dium falciparum, the causative agent of malaria. In this pathway, amplified from the corresponding genomic DNAs using a Pfu DNA polymerase which seems descended from Chlamydia bacterial pathogens with primers oXC241/242, oXC247/248, oXC249/250, oXC251/252, oXC239/240, (32), a lipoate ligase called LipL1 proceeds through the usual oXC259/oXC260, oXC233/0XC234, and oXC271/272, respectively, which added lipoyl-AMP intermediate to attach host-derived lipoate to GcvH. a ribosome binding site (5′-AAAACCATTATATTAGGAGGAAATAAC-3′)plus However, LipL1 is unable to directly attach lipoate to the SphI and HindIII restriction sites. The E. coli PDH E2 (KEGG entry EC 1.2.4.1) lipoyl domain gene was amplified from plasmid pGS331 (37, 38) using primers OADH proteins. Lipoylation of the OADH requires a second oXC235/236, which added the above ribosome binding site plus SalI and HindIII enzyme, called LipL2, to transfer lipoate from the lipoyl-AMP restriction sites. The H. sapiens sequence of the lipoyl domain of the PDH intermediate produced by LipL1 to the OADH proteins (but not E2 subunit (KEGG entry 8050), the H. sapiens gcsH (KEGG entry K02437), and to GcvH) (32). Curiously, the extant annotations indicate that P. sativum gcvH (GenBank accession no. CAJ13840) were synthesized by IDT neither P. falciparum nor Chlamydia species perform glycine cleavage. Chlamydia species lack recognizable genes that encode GcvP and GcvT subunits, whereas P. falciparum lacks GcvP encoding genes. Hence, the physiological advantage of GcvH lipoylation is unclear in these organisms and it seems that these GcvH proteins—like A. aeolicus Gcv2, Gcv3, and Gcv5—have other metabolic functions. Materials and Methods Growth Media and Conditions. Bacterial strains used in the present study are listed in Table S1. E. coli and B. subtilis strains were routinely grown in Luria Bertani (LB) broth. For complementation assays, Spizizen salts (33), supplemented with 0.5% glucose, trace elements, and 0.01% each of tryptophan and phenylalanine were used as the minimal medium for B. subtilis. Anti- biotics were added to medium at the following concentrations: sodium − − ampicillin (Amp) 100 μgmL 1; chloramphenicol (Cm) 5 μgmL 1; kanamycin −1 −1 sulfate (Km)5μgmL ; and spectinomycin sulfate (Spec) 50 μgmL . Lipoic acid (racemic) was added at 40 μM. IPTG was added at 0.5 mM. Note that the lipoic acid requirement of ΔgcvH strains can be satisfied by a mixture of Fig. 6. The phylogenetic tree of the GcvH and OADH LD obtained using the acetate and branched chain fatty acid precursors that bypass the lack of PhyML program maximum-likelihood method (34, 35). The tree is drawn to OADH activities. scale. The branch lengths are in the same units as the evolutionary distances.

Cao et al. PNAS Early Edition | 7of9 Downloaded by guest on September 30, 2021 (Integrated DNA Technologies) as gBlocks Gene Fragments and were amplified 12 h at 5 °C, and washed/eluted using buffer with 10–250 mM imidazole. The with primers oXC257 and 258, oXC231 and 232, and oXC237 and 238, re- concentrated proteins were dialyzed against 25 mM sodium phosphate buffer spectively, adding ribosome binding site plus SphI and HindIII restriction sites (pH 7.2) containing 1 mM TCEP and further purified by ion-exchange chro- as given above. Plasmids pXC.045-pXC.056 encoding GcvHs or OADH lipoyl matography using a Q column on an AFTA Purifier with increasing concen- domains were linearized and used to transform The B. subtilis ΔgcvH strain trations of NaCl. The purified proteins were then concentrated and dialyzed NM20, yielding strains XC.123-XC.129, XC.140, XC.141, XC.142, XC.147, XC.148, against 50 mM sodium phosphate (pH 7.2), 50 mM NaCl, and 1 mM TCEP. The and XC.161, respectively. Transformants were selected on plates containing B. subtilis Sfp, apo-ACP, and LipM proteins, the Vibrio harveyi AasS, and the kanamycin and spectinomycin and screened for the amyE phenotype. The E. coli AceF LD were purified as described previously (40, 41). amyE phenotype was assayed with colonies grown for 24 h in LB starch plates + by adding 10 mL of 1% I2-KI solution (39). Under these conditions, amy col- Conversion of apo-ACP to holo-ACP. The reaction mixture (100 μL) contained onies give a clear halo, whereas ΔamyE colonies produce no halo. 100 mM 2-(N-morpholino)ethanesulfonic acid (pH 6.0), 10 mM MgCl2,5mM To detect the expression levels of the proteins that failed to complement DTT, 500 μM lithium CoA, 200 μM apo-ACP, and 10 μMoftheB. subtilis Sfp growth of strain NM20, insertion of a sequence encoding the C-terminal His6 4′-phosphopantetheinyl transferase. The reaction was performed at 37 °C tag into the NM20 genomic gcvH/LD was performed. The genes that failed to for 3 h followed by incubation at 65 °C for 2 h and the precipitates were complement were PCR-amplified with a forward primer containing the above removed by centrifugation. The remaining fraction containing holo-ACP was ribosome binding site and a reverse primer encoding a His6 tag and a stop analyzed by conformationally sensitive 2 M urea PAGE (15% arylamide), and codon. E. coli gcvH, E. coli LD, H. sapiens LD, S. coelicolor LD, A. aeolicus gcvH2, then dialyzed against 25 mM sodium phosphate (pH 7.0), 300 mM NaCl, gcvH3,andgcvH5 genes were amplified with primers oXC233/277, oXC235/ 1 mM TCEP, and 10% glycerol. 278, oXC257/282, oXC259/283, oXC247/280, oXC249/281, and oXC239/279, respectively, and ligated into pDR111 to give the pXC.057 to pXC.063 plasmids. AasS/LipM Coupled Octanoyl-Transfer Reaction. The coupled reaction mixture These plasmids were then linearized and transformed into NM20 generating (25 μL) contained 100 mM sodium phosphate (pH 7.2), 50 mM NaCl, 5 mM strains XC.173-XC.179. Transformants were selected and screened for amyE disodium ATP, 5 mM TCEP, 2 mM MgCl , 0.25 mM [1-14C] sodium octanoate, phenotype as above. 2 50 μM holo-ACP, 2.5 μMoftheV. harveyi AasS acyl-ACP synthetase, 10 μM For purification of the protein products of the genes, the five A. aeolicus LipM, and ∼20 μM of one of the GcvH proteins. The reaction was performed putative gcvH genes (gcvH1, gcvH2, gcvH3, gcvH4,andgcvH5) were amplified from corresponding genomic DNA using Pfu DNA polymerase with primers at 37 °C for 5 h. The products were electrophoresed on 2 M urea PAGE (20% oHYQ188/189, oHYQ190/191, oHYQ192/193, oHYQ194/195, and oHYQ165/ arylamide), then dried under vacuum at 65 °C for 2 h and exposed to pre- − 166, respectively, which added NdeI and HindIII restriction sites. The products flashed Biomax XAR film (Kodak) at 80 °C for 24 h. were digested with NdeI and HindIII and ligated into vector pET28b to express the N-terminally hexahistidine-tagged proteins. Plasmid pGS331, which ex- Western Blot Analysis. Anti-His6 primary antibody was utilized to probe presses E. coli AceF LD under the control of the tac promoter, was the source protein expression, as described previously (42). Strains XC173 to XC179, of the E. coli AceF LD protein. The LD of H. sapiens PDH E2 and H. sapiens gcsH which encode genomic C-terminal His6-tagged versions of either a gcvH or a were PCR-amplified with NdeI and BamHI restriction sites added. The NdeI- LD, were grown to midlog phase in Spizizen salts minimum medium plus and BamHI-digested PCR fragments were ligated to pET28b generating trace elements, 0.5% (wt/vol) glucose, 0.01% tryptophan, 0.01% phenylal- pXC.043 and pXC.044, respectively. The expression vectors containing B. sub- anine, 40 μM lipoic acid, and 0.5 mM IPTG. A 2-mL aliquot of each cell culture tilis gcvH (KEGG entry BSU32800), LD of S. coelicolor branched-chain OADH E2, was harvested after 22 h of growth. Soluble fractions of whole-cell extracts and S. coelicolor gcvH were from laboratory stocks (Table S1). were analyzed by SDS/PAGE. Equal amounts of protein were loaded and separated on a 12% SDS-polyacrylamide gel and transferred by electro- Protein Purification. GcvH and LD proteins were expressed and purified from phoresis to Immobilon-P membranes (Millipore) for 30 min at 60 V. The strain BL21(DE3) (Table S1). Briefly, cultures were grown to OD600 0.5–0.8, in membranes were preblocked with TBS buffer (100 mM Tris base and 0.9% which 1 mM IPTG was added to induce protein expression. The cells were NaCl, pH 7.5) containing 0.1% Tween 20 and 5% nonfat milk powder. The harvested after 3–5 h and suspended in lysis buffer [50 mM Tris·HCl (pH 8.0), membranes were probed for 1 h with an anti-His6 protein primary antibody 300 mM NaCl, 1 mM Tris(2-carboxyethyl)phosphine (TCEP), 10 mM imidazole]. (ThermoFisher Scientific) diluted 1:2,000 in the above buffer. Following in- The cells were lysed by three passages through a French Press apparatus after cubation with a peroxidase labeled anti-mouse secondary antibody (diluted

which the insoluble matter was removed by high-speed centrifugation and 1:5,000; GE Healthcare Life Sciences), the labeled proteins (His6-tagged GcvH then filtration. Clear lysates were then incubated with Ni-NTA agarose for 3– or LD) were detected using Quantity One software.

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