Crystal Structure of Bacterial Cystathionine -Lyase in The

crystals

Article Crystal Structure of Bacterial Cystathionine Γ-Lyase in The Cysteine Biosynthesis Pathway of Staphylococcus aureus

Dukwon Lee 1 , Soyeon Jeong 1, Jinsook Ahn 1, Nam-Chul Ha 1,* and Ae-Ran Kwon 2,*

1 Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea; [email protected] (D.L.); [email protected] (S.J.); [email protected] (J.A.) 2 Department of Beauty Care Industry, College of Medical Science, Deagu Haany University, Gyeongsan, Gyeongbuk 38610, Korea * Correspondence: [email protected] (N.-C.H.); [email protected] (A.-R.K.); Tel.: +82-2-880-4853 (N.-C.H.); +82-53-819-1585 (A.-R.K.) Received: 17 October 2019; Accepted: 5 December 2019; Published: 9 December 2019

Abstract: Many enzymes require pyridoxal 5’-phosphate (PLP) as an essential cofactor and share active site residues in mediating diverse enzymatic reactions. Methionine can be converted into cysteine by cystathionine γ-lyases (CGLs) through a transsulfuration reaction dependent on PLP. In bacteria, MccB, also known as YhrB, exhibits CGL activity that cleaves the C–S bond of cystathionine at the γ position. In this study, we determined the crystal structure of MccB from Staphylococcus aureus in its apo- and PLP-bound forms. The structures of MccB exhibited similar molecular arrangements to those of MetC-mediating β-elimination with the same substrate and further illustrated PLP-induced structural changes in MccB. A structural comparison to MetC revealed a longer distance between the N-1 atom of the pyridine ring of PLP and the Oδ atom of the Asp residue, as well as a wider and more flexible active site environment in MccB. We also found a hydrogen bond network in Ser-water-Ser-Glu near the Schiff base nitrogen atom of the PLP molecule and propose the Ser-water-Ser-Glu motif as a general base for the γ-elimination process. Our study suggests the molecular mechanism for how homologous enzymes that use PLP as a cofactor catalyze different reactions with the same active site residues.

Keywords: cystathionine γ-lyases; cysteine biosynthesis; Staphylococcus aureus; pyridoxal 5’-phosphate (PLP)

1. Introduction Cysteine and methionine are sulfur-containing amino acids, which are prone to oxidization by reactive oxygen species [1]. In bacteria, these two amino acids can be interconverted by transsulfuration reactions via cystathionine, which is the common intermediate [2]. For methionine biosynthesis from cysteine in bacteria, cysteine and an activated homoserine are ﬁrst linked by cystathionine γ-synthase MetB, resulting in the formation of cystathionine [3] (Figure1a). Cystathionine is then cleaved at the β position from the cysteine by cystathionine β-lyase MetC, producing homocysteine, the precursor of methionine [4]. In the biosynthesis of cysteine, cystathionine β-synthase [5], called MccA or CysM in bacteria, mediates the C-S linkage of homocysteine and serine to produce cystathionine as the ﬁrst step. Cystathionine is then cleaved at the γ position from the homocysteine by cystathionine γ-lyase, called MccB or YhrB in bacteria, producing cysteine as the product (Figure1b). Thus, the resulting sulfur atom is transferred between cysteine and homocysteine in these processes.

Crystals 2019, 9, 656; doi:10.3390/cryst9120656 www.mdpi.com/journal/crystals Crystals 2019, 9, 656 2 of 13 Crystals 2019, 9, x FOR PEERCrystals REVIEW 2019 , 9, x FOR PEER REVIEW 2 of 14 2 of 14

(a) (a) (b) (b)

Figure 1. Two metabolicFigureFigure 1.pathwaysTwo 1. metabolicTwo ofmetabolic conversion pathways pathways ofbetween conversion of methionineconversion between methionine andbetween cysteine. methionine and cysteine.(a) The and (a) Thecysteine. methionine (a) The methionine synthesissynthesis pathwaymethionine pathway from synthesis cysteine. from cysteine.pathway O-acetyl from O-acetylor O-succinylhomoserine cysteine. or O-succinylhomoserine O-acetyl or O-succinylhomoserineand cysteine and cysteineare areand linked cysteine by are linked by cystathioninecystathioninelinked γ-synthase by γcystathionine-synthase (CGS). The (CGS). γR--synthasegroup The R-group is (CGS).acetyl is or acetylThe succinyl. R-group or succinyl. Cystathionine is acetyl Cystathionine or succinyl. β-lyaseβ-lyase Cystathionine (CBL) cleaves β-lyase (CBL) cleaves the bondthe bond(CBL)between between cleaves β-carbonβ the-carbon andbond sulfur, and between sulfur, which whichβ -carbonis indicated is indicated and by sulfur, “ by “ ⸽ ”which in the figure.is indicated Homocysteine, by “⸽” in a productthe figure. of CBL,Homocysteine, is finally converted a product into of methionineCBL, is finally by anotherconverted enzyme, into methionine which is not by shown another in thisenzyme, figure. which (b) is Cysteinenot shown synthesis in this pathway figure. from (b) methionine.Cysteine synthesis Homocysteine, pathway which from ismethionine. made from Homocysteine, methionine, is linked which is figure. Homocysteine,with madea serine, product from resulting ofmethionine, CBL, in is cystathionine finally is linked converted wi byth serine, the into action methionineresulting of cystathionine in bycystathionin anotherβ-synthase enzyme,e by the (CBS).action Synthesizedof cystathionine which is not showncystathionine in βthis-synthase figure.is (b(CBS). cleaved) Cysteine Synthesized between synthesis the cyst pathwayγ-carbonathionine from and is sulfurmethionine. cleaved by cystathioninebetween Homocysteine, theγ -lyaseγ-carbon (CGL), and makingsulfur by which is made fromα -ketobutyrate methionine,cystathionine is and linkedγ-lyase cysteine with(CGL), as serine, products. making resulting α-ketobutyrate in cystathionine and cysteine by the as actionproducts. of cystathionine β-synthase (CBS). Synthesized cystathionine is cleaved between the γ-carbon and MetB,sulfur by MetC, cystathionine and MetB,MccB MetB,γ-lyase MetC,are MetC, homologous(CGL), and andmaking MccB MccB and areα-ketobutyrate are homologousshare homologous a common and and cysteine and shareevolutionary share as a products. common a common ancestor evolutionary evolutionary [6]. They ancestor ancestor [6]. [6]. They They are all PLP-dependentareare all enzymes, PLP-dependentall PLP-dependent forming enzymes, a homotetramer enzymes, forming forming with a homotetramer a each homotetramer individual with withhomodimer each each individual individual to create homodimer homodimer to createto create two active sites [7]two. Atwo activePLP active molecule sites sites [7]. is[7]. A covalently PLP A PLP molecule molecule linked is covalentlyto is thecovalently amine linked moiety linked to theofto thethe amine activeamine moiety site moiety lysine of theof the active active site site lysine lysine residue via a Schiffresidue baseresidue bond, via via a resulting Schi a Schiffff base inbasebond, an bond,internal resulting resulting aldimine in an in internalstructure.an internal aldimine When aldimine the structure. Schiff structure. base When Whenbond the the Schi Schiffff base base bond bond with PLP is movedwith towith PLPthe PLPsubstrate is moved is moved amine to the to substratemoietythe substrate in amine the amineexternal moiety moiety aldimine in the in external the structure, external aldimine the aldimine free structure, active structure, the free the active free siteactive site lysine residuelysine firstsite residueplays lysine a firstresiduegeneral plays firstacid/base a general plays rolea acid general in/base the roleacid/basedeprotonation in the role deprotonation in of the the deprotonation α-carbon of the α of-carbon the of the of theα-carbon substrate. of the substrate. DespiteDespite thesubstrate. similar the similarthree Despite-dimensional three-dimensional the similar struct three-dimensionalures structures and arrangements and stru arrangementsctures of and similar arrangements of active similar site active of similar site residues, active site residues, their activitiestheirresidues, activities differ. theirIn di particular,ff activitieser. In particular, MetCdiffer. and In MetC particular, MccB and share MccB MetC theshare substrate, and MccB the substrate, cystathionine, share the cystathionine, substrate, and cystathionine, and contain and contain different di cleavageffcontainerent cleavage sites.different While sites. cleavage MetCWhile MetC exhibitssites. exhibitsWhile β-elimination βMetC-elimination exhibits activity, activity, β-elimination MccB MccB is primarily is activity, primarily MccB responsible is primarily for γ responsible for thethe γresponsible--eliminationelimination for process process the γ and-elimination and can can also also produceprocess produce Hand2 S[ H 8can2,S9 ].[8,9] Althoughalso. Althoughproduce molecular H molecular2S [8,9]. mechanisms Although have molecular been mechanisms havesuggested beenmechanisms suggested for these havefor these enzymes, been enzymes, suggested the detailedthe for detailed these residues enzymes, residues that ththat acte detailed act as theas the general residues general acid that acid or act base as the in general catalysis acid or base in catalysisremain remainor base to to be in be elucidated.catalysis elucidated. remain to be elucidated. Staphylococcus aureusStaphylococcus Staphylococcusis an important aureus aureus humanis anis importantan pathogen important humanthat human can pathogen infect pathogen a wide that that canrange can infect ofinfect human a wide a wide range range of humanof human tissues, such as thetissues, gastrointestinaltissues, such such as the as tract, the gastrointestinal gastrointestinal heart, and bones tract, tract, [10] heart, .heart, The and m andetabolism bones bones [10 ]. [10].of The this The metabolism pathogen metabolism may of thisof this pathogen pathogen may may be linked to staphylococcalbe linkedbe linked tovirulence staphylococcalto staphylococcal factor synthesis, virulence virulence which factor factor has synthesis, synthebecomesis, which a majorwhich has research has become become topic a major a [11]major research. research topic topic [11 [11].]. In S. aureus, cystathionineIn S.In aureus, S. aureus, β-synthase cystathionine cystathionine MccA βor-synthase CysMβ-synthase (locus MccA MccA tag: or SAV0459) CysMor CysM (locus (locusand tag: cystathionine ta SAV0459)g: SAV0459) andγ-l andyase cystathionine cystathionine γ-lyase γ-lyase MccB or YhrB (locusMccB tag:MccB or SAV0460, YhrBor YhrB (locus referred(locus tag: tag: SAV0460, to SAV0460,as SaMccB referred referred in this to asstudy) to SaMccBas SaMccB are located in this in this study)in thestudy) gene are are located cluster located in the in the gene gene cluster cluster that produces cysteine.thatthat produces In produces the present cysteine. cysteine. study, In theIn we the present determined present study, study, the we crystal we determined determined structure the ofthe crystal SaMccB crystal structure structurein the of SaMccBof SaMccB in thein the absence and presenceabsenceabsence of bound and and presence PLP presence (these of bound areof bound the PLPfirst PLP (thesebacterial (these are cystathionineare the the first first bacterial bacterial γ-lyase cystathionine cystathionine structuresγ). -lyaseWe γ-lyase structures). structures). We We propose candidatepropose residuespropose candidate for candidate the residuesgeneral residues acid/base for the for general the roles general acidbased/base acid/base on roles a structural based roles onbased comparison a structural on a structural comparisonwith comparison with MetC. with MetC. MetC.

2. Materials and Methods2. Materials and Methods

Crystals 2019, 9, 656 3 of 13

2. Materials and Methods

2.1. Cloning, Protein Expression, and Purification S. aureus subsp. Mu50 yhrB gene (SAV0460) was inserted in expression vector pLIC-His using a ligation independent cloning method, as previously described [12,13]. The resulting construct had 17 additional residues (MHHHHHHENLYFQGAAS) that encoded an N-terminal hexa-histidine tag and a TEV cleavage site. Following TEV cleavage, the protein retained four additional residues (GAAS) at the N-terminus. Recombinant plasmid pLIC-SaMccB was transformed into the Escherichia coli BL21(DE3) expression host (Novagen, Madison, WI, USA). The cells were cultured in 1.5 L LB medium supplemented with 100 µg/mL ampicillin at 37 ◦C until the OD600 reached 0.6, and then 0.5 mM isopropyl-β-D-thiogalactoside was added to the medium to induce the expression of the SaMccB protein. After further culturing for 6 h at 30 ◦C, the cells were harvested by centrifugation and resuspended in 50 mL of lysis buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 2 mM β-mercaptoethanol. The resuspended cells were disrupted by sonication, and the cell debris was removed by centrifugation at 20,000 g for 30 min at 4 C. The His-tagged SaMccB protein was × ◦ purified using Ni-NTA agarose resin (2 mL; Qiagen, Hilden, Germany). After loading the protein, the resin was washed with 500 mL of wash buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 20 mM imidazole (pH 8.0), and 2 mM β-mercaptoethanol. The SaMccB protein was eluted with 15 mL of elution buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 250 mM imidazole (pH 8.0), and 2 mM β-mercaptoethanol. The TEV enzyme was treated at 20 ◦C overnight to eliminate non-SaMccB peptides. Q anion exchange chromatography (5 mL HiTrap Q HP; GE Healthcare Life Sciences, Pittsburg, PA, USA) was used for purification with a gradient of NaCl (0–1 M). The protein was eluted in a range of 300–400 mM NaCl, and the pooled fractions were concentrated to 500 µL using a Vivaspin centrifugal concentrator (30 kDa molecular weight cutoff; Millipore, Hayward, CA, USA), and diluted to 5 mL with a buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 2 mM β-mercaptoethanol. The final concentration of the protein was 15 mg/mL, which was stored at 80 ◦C until use.

2.2. Crystallization SaMccB crystallization trials with and without cofactor PLP were performed using an automated ® crystal screening device (Mosquito Crystal; TTP Labtech, Melbourn, UK) at 14 ◦C in a sitting-drop vapor-diﬀusion plate (Axygen™; Axygen, Union City, CA, USA). The HR2-082, 084, 098, 110, 112, and 126 screening solutions (Hampton Research, Aliso Viejo, CA, USA) were used as reservoir solutions. Square bipyramid-shaped crystals of SaMccB without PLP were observed in the reservoir solution drop consisting of 8% (v/v) Tacsimate (pH 7.0) and 20% PEG 3350 (Supplementary Materials Figure S1a). The crystallization conditions were optimized with the EasyXtal 15-well Tools (Qiagen, Hilden, Germany) in the reservoir solution drop consisting of 10% (v/v) Tacsimate (pH 7.0) and 20% PEG 3350. For PLP-bound SaMccB, the SaMccB solution containing 400 µM PLP was initially crystallized in a reservoir solution containing 0.1 M sodium acetate (pH 4.0) and 30% PEG 300 and then optimized in a solution containing 0.1 M sodium acetate (pH 5.0) and 28% PEG 400 (Supplementary Materials Figure S1b).

2.3. Structural Determination The X-ray diffraction datasets of apo- and PLP-bound SaMccB crystals were collected on an EIGER-9M detector (DECTRIS, Baden-Daettwil, Switzerland) in a beamline 5C from the Pohang Accelerator Laboratory (Pohang, Republic of Korea) at a wavelength of 1.00003 Å. Crystals for apo-SaMccB were soaked for 5 s in a cryoprotectant buffer containing an additional 25% glycerol in its own reservoir solution and then flash-cooled in liquid nitrogen at 173 C. For PLP-bound SaMccB − ◦ crystals, their own reservoir solution was used for the cryoprotectant. The diffraction datasets were processed by the HKL-2000 program [14], and the molecular replacement method was performed using Crystals 2019, 9, 656 4 of 13 the CCP4 program, with the Xanthomonas oryzae MetB (XoMetB) structure (PDB code 4L0O; sequence identity 54.1%) as a search model. The structure of the apo-SaMccB was determined and refined using Coot and Phenix [15,16]. The PLP-bound SaMccB structure was solved using the apo-MccB structure in the molecular replacement method. The statistics for data collection and model refinement are summarized in Table1.

Table 1. X-ray data statistics and reﬁnement of the structure.

Apo-SaMccB PLP-bound SaMccB Data collection Beam line PAL5C PAL5C Wavelength (Å) 1.00003 1.00003 Space group I4122 P42212 Cell dimensions a, b, c (Å) 105.567, 105.567, 287.986 132.281, 132.281, 97.954 α, β, γ (◦) 90, 90, 90 90, 90, 90 Resolution (Å) 50.0–2.30 (2.34–2.30) 50.0–2.30 (2.34–2.30) Rmerge 0.121 (0.415) 0.070 (0.349) Rpim 0.030 (0.159) 0.017 (0.126) I/σI 16.7 (2.4) 28.11 (3.92) Completeness (%) 99.6 (99.6) 99.5 (98.7) Redundancy 14.7 (6.2) 14.1 (7.5) Wilson B-factor (Å2) 25.9 24.3 Reﬁnement Resolution (Å) 46.59–2.30 42.21–2.30 No. reﬂections 33271 38561 Rwork/Rfree 0.202/0.220 0.203/0.249 Number of total atoms 2867 5828 Average B-factor (Å2) All atoms 30.0 30.0 Lysine-PLP ND 20.89 Solvent atoms 25.56 22.94 R.M.S deviations Bond lengths (Å) 0.002 0.002 Bond angles (◦) 0.471 0.491 Ramachandran plot Favored (%) 96.7 97.0 Allowed (%) 3.0 3.0 Outliers (%) 0.3 0.0 PDB code 6KGZ 6KHQ

PLP, pyridoxal 5’-phosphate. Rmerge = Σ Σ |I (hkl) [I(hkl)]|/Σ Σ I (hkl), where I (hkl) is the intensity of the ith hkl i i − hkl i i i observation of reﬂection hkl and [I(hkl)] is the average intensity of i observations. R = Σ (1/(n 1))1/2 Σ |I (hkl) p.i.m. hkl − i i [I(hkl)]|/Σ Σ I (hkl). R is the precision-indicating (multiplicity-weighted) Rmerge. − hkl i i pim

2.4. Multi-Angle Light Scattering Analysis For high-performance liquid chromatography (Shimadzu, Kyoto, Japan), a Superdex 75 Increase 10/300 GL column (GE Healthcare Life Sciences, Pittsburgh, PA, USA), equipped with a multi-angle light scattering (MALS) instrument (Wyatt DAWN Heleos II (18 angles); Wyatt Optilab T-rex (RI); Wyatt Technology, Goleta, CA, USA) was used at the KBSI Ochang Center (Ochang, Republic of Korea). The protein sample (2 mg/mL) was applied to the column in a buﬀer containing 20 mM Tris (pH 8.0) and 150 mM NaCl. Data analyses were performed using the ASTRA 6 software (Wyatt Technology, Goleta, CA, USA). Crystals 2019, 9, 656 5 of 13 Crystals 2019, 9, x FOR PEER REVIEW 5 of 14

(pH 8.0) and 150 mM NaCl. Data analyses were performed using the ASTRA 6 software (Wyatt 2.5. Data Availibility Technology, Goleta, CA, USA). The atomic coordinates and structure factor ﬁles of the apo- and PLP-bound SaMccB structures 2.5. Data Availibility described in this research have been deposited in the Protein Data Bank (www.wwpdb.org) with the accession codesThe 6 atomic KGZ andcoordinates 6 KHQ, and respectively. structure factor files of the apo- and PLP-bound SaMccB structures described in this research have been deposited in the Protein Data Bank (www.wwpdb.org) with the 3. Resultsaccession codes 6 KGZ and 6 KHQ, respectively.

3.1. Structural3. Results Determination of SaMccB The monomeric3.1. Structural structure Determination of SaMccB of SaMccB shows overall folds similar to type I PLP-dependent enzymes, including humanThe CGL,monomeric MetB, structure and MetC of SaMccB [4,6] (Figure shows2 a).overall The puriﬁedfolds similar SaMccB to type protein I PLP-dependent (monomer size, 42 kDa) wasenzymes, a homotetramer including human (~160 CGL, kDa) MetB, in a solution, and MetC as [4,6] determined (Figure 2a). using The SEC-MALSpurified SaMccB (Supplementary protein Materials Figure(monomer S2). size, Consistent 42 kDa) was with a thehomotetramer SEC-MALS (~160 results, kDa) bothin a solution, the crystal as determined structures ofusing SaMccB SEC- with MALS (Supplementary Materials Figure S2). Consistent with the SEC-MALS results, both the crystal and without PLP binding showed that SaMccB was a homotetramer constructed as a dimer of dimers. structures of SaMccB with and without PLP binding showed that SaMccB was a homotetramer The asymmetricconstructed unit as of aapo-SaMccB dimer of dimers. contained The asymmetric one protomer unit of apo-SaMccB in the I4122 contained space group, one protomer and the in tetramerthe was formedI4122 by space the crystallographicgroup, and the tetramer symmetry was formed in the by crystal the crystallographic (Figure2b). Thesymmetry PLP-bound in the crystal structure contained(Figure a dimer 2b). in The the PLP-bound asymmetric structure unit in containedP42212, anda dimer the in tetramer the asymmetric was formed unit in byP42 crystallographic212, and the 2-fold symmetrytetramer (Figurewas formed2c). by crystallographic 2-fold symmetry (Figure 2c).

(a)

(b)

Figure 2. Cont. Crystals 2019, 9, 656 6 of 13 Crystals 2019, 9, x FOR PEER REVIEW 6 of 14

(c)

Figure 2. TetramericFigure 2. Tetrameric assembly assembly of cystathionine of cystathionineγ-lyase γ-lyase MccB MccB or or YhrBYhrB (SaMccB). (a) ( aThe) The monomeric monomeric unit of the apo-SaMccB structure shown in the ribbon diagram. The secondary structural elements unit of the apo-SaMccB structure shown in the ribbon diagram. The secondary structural elements are are labeled in order of sequence number. α-helixes are colored in cyan, β-strands are colored in α β labeled in ordermagenta, of and sequence loops are number. colored in-helixes orange. Sequences are colored that in are cyan, not shown-strands in the are structure colored are in drawn magenta, and loopsas are a coloreddotted line. inorange. (b) The Sequencestetrameric unit that of arethe notapo-SaMccB shown instructure. the structure The asymmetric are drawn unit as (one a dotted line. (b) Theprotomer) tetrameric of the apo-SaMccB unit of the structure apo-SaMccB is colored structure. in brown The(N-terminal asymmetric domain), unit green (one [pyridoxal protomer) 5'- of the apo-SaMccBphosphate structure (PLP)-binding is colored domain], in brown or teal blue (N-terminal (C-terminal domain), domain). greenThe remaining [pyridoxal three 5’-phosphatesubunits, (PLP)-bindinggenerated domain], by the orsymmetry teal blue operation, (C-terminal are in grey. domain). (c) The The tetrameric remaining unit of three the PLP-bound subunits, SaMccB generated structure. One protomer in the asymmetric unit is colored in cyan (N-terminal domain), orange (PLP- by the symmetry operation, are in grey. (c) The tetrameric unit of the PLP-bound SaMccB structure. binding domain), and magenta (C-terminal domain). The other protomer in the asymmetric unit is One protomercolored in in the lime. asymmetric The boundunit PLP ismolecules colored are in repr cyanesented (N-terminal by sticks. domain), The remaining orange two (PLP-binding subunits, domain), andgenerated magenta by the (C-terminal symmetry operation, domain). are The in grey. other protomer in the asymmetric unit is colored in lime. The bound PLP molecules are represented by sticks. The remaining two subunits, generated by the symmetryEach operation, subunit consisted are in grey. of three domains: an N-terminal domain (residues 1–50), a PLP-binding domain (residues 51–230), and a C-terminal domain (residues 231–380) (Figure 2b). The N-terminal Eachdomain subunit was consisted composed of threeof one domains:α-helix (α1, an residues N-terminal 3–9) and domain one long (residues loop interacting 1–50), a PLP-bindingwith the domain (residuesneighboring 51–230), subunit, and which a C-terminal was involved domain in dimerization. (residues The 231–380) PLP-binding (Figure domain2b). Theconsisted N-terminal of parallel and antiparallel seven-stranded β-sheets (β1, residues 68–72; β2, residues 91–95; β3, residues domain was composed of one α-helix (α1, residues 3–9) and one long loop interacting with the 116–120; β4, residues 136–142; β5, residues 167–171; β6, residues 189–193; and β7, residues 207–211) neighboringin the subunit, core, with which neighboring was involved α-helices ( inα2, dimerization. residues 51–63; α The3, residues PLP-binding 75–83; α4, domain residues 100–108; consisted of parallel andα5, antiparallel residues 126–131; seven-stranded α6, residues β154–163;-sheets α (β7,1, residues residues 176–179; 68–72; andβ2, α residues8, residues 91–95; 214–227).β3, This residues 116–120; βdomain4, residues contained 136–142; the mostβ5, residuesactive site 167–171; residues βand6, residuescovalently 189–193; linked Lys196 and β to7, the residues PLP molecule 207–211) in the core, with(Lys210 neighboring in XoMetC).α The-helices C-terminal (α2, residues domain was 51–63; structuredα3, residues into five-stranded 75–83; α4, antiparallel residues β 100–108;-sheets α5, (β8, residues 273–274; β9, residues 297–301; β10, residues 319–320; β11, residues 330–332; and β12, residues 126–131; α6, residues 154–163; α7, residues 176–179; and α8, residues 214–227). This domain residues 356–360) decorated with α-helices (α9, residues 233–243; α10, residues 246–264; α11, containedresidues the most 284–290; active α site12, residues 305–314; and covalently α13, residues linked 334–337; Lys196 α14, to residues the PLP 344–350; molecule and (Lys210 α15, in XoMetC). Theresidues C-terminal 366–377). domain The PLP was binding structured and C-terminal into five-stranded domains were antiparallel both cystathionineβ-sheets β-synthase (β8, residues 273–274; β(CBS)9, residues domains, 297–301; formingβ a10, CBS residues pair or Bateman 319–320; doβmain11, residues [17]. Like 330–332; the usual and Batemanβ12, domain, residues these 356–360) decoratedtwo with domainsα-helices surrounded (α9, residues the active 233–243; site pocketα of10, SaMccB. residues 246–264; α11, residues 284–290; α12, residues 305–314; α13, residues 334–337; α14, residues 344–350; and α15, residues 366–377). The PLP 3.2. The Active Site and the Ser–Water–Ser–Glu Network binding and C-terminal domains were both cystathionine β-synthase (CBS) domains, forming a CBS pair or BatemanThe domain active site [17 of]. SaMccB Like thewas usual lined with Bateman residues domain, from the these PLP-binding two domains domain and surrounded residues the of the N-terminal domain in the neighboring subunit. Most residues at the active site were shared active site pocket of SaMccB. with human CGL, MetB, and MetC (Figure 3a). When superimposed with the XoMetC structure in the complex with PLP, subtle conformational differences were found. Tyr99 in the β-strand of the 3.2. The Active Site and the Ser–Water–Ser–Glu Network PLP-binding domain and Tyr45 in the N-terminal domain of the neighboring subunit were notable The activein the active site of site SaMccB (Figure 3b). was Tyr99 lined made with the residues pi-interaction from with the the PLP-binding pyridine ring domain of PLP in and SaMccB, residues while the corresponding residue (Tyr112) in XoMetC largely interacted with the conjugated double of the N-terminal domain in the neighboring subunit. Most residues at the active site were shared bonds around the Schiff base nitrogen (NSB) of PLP (Figure 3b). with human CGL, MetB, and MetC (Figure3a). When superimposed with the XoMetC structure in the complex with PLP, subtle conformational differences were found. Tyr99 in the β-strand of the PLP-binding domain and Tyr45 in the N-terminal domain of the neighboring subunit were notable in the active site (Figure3b). Tyr99 made the pi-interaction with the pyridine ring of PLP in SaMccB, while the corresponding residue (Tyr112) in XoMetC largely interacted with the conjugated double bonds around the Schiff base nitrogen (NSB) of PLP (Figure3b). Crystals 2019, 9, x FOR PEER REVIEW 7 of 14

Asp171 or its equivalent residue is known to stabilize the positive charge of N1 in the pyridine ring of PLP in PLP-dependent enzymes [9]. A structural comparison with the MetC structure in the internal aldimine showed that the distance between the Asp171 residue and the N1 atom of PLP in SaMccB was longer than that in the MetC structure. Instead, the Ser193 residue was closer to the phosphate of PLP, compared to that of MetC (Figure 3b). Importantly, a strong electron density map indicated that a water molecule was found between Ser323 and Ser202, whose hydrogen bonds were then extended to Glu328 buried in the hydrophobic core region (Figure 3c). The Oγ of Ser323 was near the Cδ of Lys linked to PLP. All three residues are Crystals 2019 9 strictly ,conserved, 656 among type I PLP-dependent enzymes (Figure 3a), indicating their functional 7 of 13 importance.

Crystals 2019, 9, x FOR PEER REVIEW 8 of 14 (a)

(b)

Figure 3. Cont.

(c)

Figure 3. Local environment at the active site. (a) Sequence alignment of SaMccB, human CGL (PDB code 2NMP), Helicobacter pylori CGS (PDB code 4L0O; HpCGS), and Xanthomonas oryzae MetC (PDB code 4IXZ; XoMetC). Identical residues are colored white on a red background, and similar residues are red on a yellow background. Secondary structural elements (springs, α-helices, arrows, and β- strands) are represented above the sequences and are numbered. The red circles indicate the active site residues. The figure was constructed using ESPript (http://espript.ibcp.fr) [18]. (b) Active site residues of the pyridoxal 5'-phosphate (PLP)-bound SaMccB structure (up) compared with the PLP- bound structure of XoMetC (down). Both structures are shown in stereo images. Color codes for Crystals 2019, 9, x FOR PEER REVIEW 8 of 14

Crystals 2019, 9, 656 8 of 13

(b)

(c)

FigureFigure 3. Local 3. Local environment environment at at the the active active site.site. (a) (a) Sequence Sequence alignment alignment of SaMccB, of SaMccB, human human CGL CGL(PDB (PDB codecode 2NMP), 2NMP),Helicobacter Helicobacter pylori pyloriCGS CGS (PDB(PDB code 4L0O; 4L0O; HpCGS), HpCGS), and and XanthomonasXanthomonas oryzae oryzae MetCMetC (PDB (PDB code 4IXZ; XoMetC). Identical residues are colored white on a red background, and similar residues code 4IXZ; XoMetC). Identical residues are colored white on a red background, and similar residues are are red on a yellow background. Secondary structural elements (springs, α-helices, arrows, and β- red on a yellow background. Secondary structural elements (springs, α-helices, arrows, and β-strands) strands) are represented above the sequences and are numbered. The red circles indicate the active are represented above the sequences and are numbered. The red circles indicate the active site residues. site residues. The figure was constructed using ESPript (http://espript.ibcp.fr) [18]. (b) Active site Theresidues ﬁgure was of the constructed pyridoxal 5'-phosphate using ESPript (PLP)-bound (http://espript.ibcp.fr SaMccB structure)[18 ].(up) (b )compared Active site with residues the PLP- of the pyridoxalbound 5’-phosphatestructure of XoMetC (PLP)-bound (down). SaMccB Both structur structurees are (up) shown compared in stereo with images. the PLP-bound Color codes structure for of XoMetC (down). Both structures are shown in stereo images. Color codes for SaMccB are the same as in Figure2c. The N-terminal domain of another subunit of XoMetC is colored in yellow, the PLP-binding domain of XoMetC is in purple, and PLP is in pink. The distances between PLP and the surrounding interacting residues are indicated. (c) The Ser–water–Ser–Glu hydrogen bond network positioned near PLP. Ser323 is near the Schiﬀ base nitrogen atom of the PLP molecule (3.5 Å). The hydrogen bond network is indicated by the red dotted lines. The simulated annealing OMIT map was generated without the PLP-linked Lys and solvent (black, the Fo –Fc map contoured at 3.0 σ). Color codes are the same as in Figure2c, except PLP is colored in pink.

Asp171 or its equivalent residue is known to stabilize the positive charge of N1 in the pyridine ring of PLP in PLP-dependent enzymes [9]. A structural comparison with the MetC structure in the internal aldimine showed that the distance between the Asp171 residue and the N1 atom of PLP in SaMccB was longer than that in the MetC structure. Instead, the Ser193 residue was closer to the phosphate of PLP, compared to that of MetC (Figure3b). Importantly, a strong electron density map indicated that a water molecule was found between Ser323 and Ser202, whose hydrogen bonds were then extended to Glu328 buried in the hydrophobic core region (Figure3c). The O γ of Ser323 was near the Cδ of Lys linked to PLP. All three residues are strictly conserved among type I PLP-dependent enzymes (Figure3a), indicating their functional importance.

3.3. Structural Flexibility Depending on PLP Binding To analyze the structural changes upon PLP binding, we superimposed the apo-MccB onto the PLP-bound MccB structures, which showed that the overall structures were similar (rmsd of 0.375 Å between the 317 Cα atoms). However, we found two regions that were noticeably different between the two structures. Upon PLP binding to Lys196, the loop (residues Ile36–Thr48) from the N-terminal domain in the neighboring subunit became ordered, while the α-helical flap (α13–α14 and their flanking loop, residues Val333–Glu350) of the C-terminal domain became more flexible (Figure4b,c). In the apo-MccB structure, the conserved Tyr99 at the active site of hydrogen bonded with His339 and Ser341 of the α-helical flap region, presumably stabilizing the α-helical flap (Figure4a). However, these hydrogen bonds with His339 and Ser341 were broken by PLP binding because Tyr99 was moved for the pi-interaction with the pyridine ring of PLP (Figure4b,c). Crystals 2019, 9, x FOR PEER REVIEW 9 of 14

SaMccB are the same as in Figure 2c. The N-terminal domain of another subunit of XoMetC is colored in yellow, the PLP-binding domain of XoMetC is in purple, and PLP is in pink. The distances between PLP and the surrounding interacting residues are indicated. (c) The Ser–water–Ser–Glu hydrogen bond network positioned near PLP. Ser323 is near the Schiff base nitrogen atom of the PLP molecule (3.5 Å). The hydrogen bond network is indicated by the red dotted lines. The simulated annealing

OMIT map was generated without the PLP-linked Lys and solvent (black, the Fo – Fc map contoured at 3.0 σ). Color codes are the same as in Figure 2c, except PLP is colored in pink.

3.3. Structural Flexibility Depending on Plp Binding To analyze the structural changes upon PLP binding, we superimposed the apo-MccB onto the PLP-bound MccB structures, which showed that the overall structures were similar (rmsd of 0.375 Å between the 317 Cα atoms). However, we found two regions that were noticeably different between the two structures. Upon PLP binding to Lys196, the loop (residues Ile36–Thr48) from the N-terminal domain in the neighboring subunit became ordered, while the α-helical flap (α13–α14 and their flanking loop, residues Val333–Glu350) of the C-terminal domain became more flexible (Figure 4b,c). In the apo-MccB structure, the conserved Tyr99 at the active site of hydrogen bonded with His339 and Ser341 of the α-helical flap region, presumably stabilizing the α-helical flap (Figure 4a). However, these hydrogen bonds with His339 and Ser341 were broken by PLP binding because Tyr99 was moved for the pi-interaction with the pyridine ring of PLP (Figure 4b,c). Unlike SaMccB, an increase in the structural flexibility of the α-helical flap region was not observed in the MetC structure. The α-helical flap region of the MetC structure was fixed by the Crystalsinteraction2019, 9of, 656 Arg362 and the backbone carbonyl group of Asp109 in the PLP-binding domain,9 of 13 regardless of PLP binding (Figure 4).

(a) (b)

Figure 4. Changes in structural flexibilityflexibility upon pyridoxal 5’-phosphate5'-phosphate (PLP) binding. ( a) Active site configurationsconfigurations of apo-SaMccB.apo-SaMccB. Tyr99 interacts with His339 and Ser341, which renders the α-helical-helical flap (residues Val333–Glu350), consisting of α13–α14, stable and rigid. By contrast, the loop (residues

Ile36–Thr48) from the N-terminal domain of the adjacent subunit is disordered. The α-helical flap is indicated by the black arrow and box. Color codes are the same as in Figure2b. ( b) Active site configurations of PLP-bound SaMccB. Tyr99 interacts with the PLP pyridine ring and Tyr45 and Arg47 from the loop of the N-terminal domain of the adjacent subunit interaction with the phosphate region. The α-helical flap region is disordered, likely due to lack of Tyr99-mediated interactions. Color codes are the same as in Figure2c. ( c) Superimposed structure image of apo-SaMccB and PLP-bound SaMccB. Tyr99 shows a large difference in position and makes the changes indicated above. Color codes are same as in Figure2b,c. ( d) Active site configurations of the PLP-bound XoMetC. The Arg362 of the α-helical flap region facilitates interaction with the backbone carbonyl group of Asp109 in the PLP-binding domain, stabilizing the conformation of the α-helical flap region despite the lack of Tyr112 (Tyr99 in the SaMccB numbering)-mediated interaction. Color codes are the same as in Figure3b, down.

Unlike SaMccB, an increase in the structural flexibility of the α-helical flap region was not observed in the MetC structure. The α-helical flap region of the MetC structure was fixed by the interaction of Arg362 and the backbone carbonyl group of Asp109 in the PLP-binding domain, regardless of PLP binding (Figure4).

4. Discussion This study showed the crystal structures of MccB from S. aureus in the presence and absence of PLP at the active site. The overall structures of the bacterial MccB shared structural organization with human CGL and its homologues, MetB and MetC, in bacteria (Supplementary Materials Figure S3). A structural Crystals 2019, 9, 656 10 of 13 comparison of the apo- and PLP-bound structures of SaMccB showed that the inter-subunit loop and the α-helical flap region have substantially different conformational constraints. The PLP-bound structures became flexible in the α-helical flap region; this was different from MetC, which showed a stable interaction regardless of PLP binding (Figure4b,d). Delocalized electrons by conjugated double bonds and the electron sink at the N1-atom of PLP are essential for most PLP-mediated reactions. The Lys residue for PLP binding is commonly used as the first and primary general acid/base when it is free from PLP binding. However, the other general acid/base at the active site residues might also be important in determining which reactions are preferred in the individual enzyme. By comparing the reaction processes between the γ-elimination of MccB and the β-elimination of MetB (Figure5), we identified the importance of the second base residue (Base in Figure5b) for deprotonation at the β-carbon in MccB, which is a prerequisite for γ-elimination. The resulting carbanion at Cβ was stabilized by NSB through a new pi-bond between Cβ and Cγ. We noted a wider active site of SaMccB, which might prefer deprotonation at Cβ. Because the NSB atom of PLP turned from sp2 to sp3 orbital, and the pseudo-planar conjugated double bond system to the pyridine ring was broken at NSB, more room was required to hold this intermediate. The structural flexibility of MccB could better compensate for this conformational change during the γ-elimination reaction. What would be the second base residue in MccB? We noted the Ser–water–Ser–Glu hydrogen bond network at the active site of MccB. According to the model structure of the external aldimine (step II in Figures5b and3c), O γ of Ser323 was near the Cβ atom of the Schiff base nitrogen atom of PLP, within 3.5 Å. Glu328 was surrounded in a hydrophobic environment and was connected to Ser323 via Ser202 and the water molecule (Figure3c). Because the pKa value of Glu328 increased due to its hydrophobic environment, the proton of Ser328 was easily transferred in this network. This hydrogen bond network was comparable with the catalytic triad, Ser–His–Asp, in traditional serine proteases, where the proton of Ser is transferred to Asp under a hydrophobic environment via His. S. aureus Mu50 (ATCC 700699) is a vancomycin intermediate S. aureus that displays robust virulence properties. It causes severe infectious diseases ranging from mild infections, such as skin infections and food poisoning, to life-threatening infections, such as sepsis, endocarditis, and toxic shock syndrome [19]. However, because of bacterial resistance, the prognosis for S. aureus infection is still poor despite early diagnosis and appropriate treatment [20]. Although there are various theories regarding how S. aureus acquires its antibiotic resistance, very little is known. In this regard, understanding the bacterial defense mechanisms of S. aureus against antibiotics is crucial. Many bactericidal antibiotics kill the bacteria by stimulating the production of highly toxic hydroxyl radicals, whose production is mediated by Fe2+ [21]. To cope with this stress, the bacteria could alter gene expression levels and metabolism, which are possibly linked to staphylococcal virulence factor synthesis and inhibition of the production of hydroxyl radicals [11]. SaMccB (named YhrB in the reference) is commonly listed as a gene that is downregulated by ampicillin, kanamycin, and norfloxacin. Because free cysteine could accelerate the Fe2+-mediated Fenton reaction by reducing Fe3+ to Fe2+ [22], the downregulation of SaMccB in response to diverse antibiotics could be explained. To control the resistance of S. aureus, a better understanding of metabolic enzymes is important. Our study provided molecular clues into how homologous enzymes using PLP as the cofactor catalyze different reactions of metabolic enzymes, whose gene expression is regulated by antibiotic stress. Further research will promote a better understanding of how bacteria can defend against conditions of stress. Crystals 2019, 9, 656 11 of 13 Crystals 2019, 9, x FOR PEER REVIEW 11 of 14

(a)

(b)

Figure 5. ComparisonComparison of of the the enzymatic enzymatic me mechanismschanisms between between cystathionine β-lyase-lyase MetC MetC and cystathionine γ-lyase-lyase MccB. ((a)a) CystathionineCystathionine β-lyase MetC. 1.1. Internal Internal aldimine: The The Schiff Schiﬀ base linkagelinkage of of pyridoxal 5'-phosphate 5’-phosphate (PLP) (PLP) to to the the en enzymezyme Lys Lys residue (interna (internall aldimine) is exchanged with the the amine amine group group of of the the cystei cysteinene of of cystathionine cystathionine (external (external aldi aldimine).mine). 2. External 2. External aldimine: aldimine: The protonThe proton at the at α the-carbonα-carbon of the of substrate the substrate is abstracted is abstracted by the by NZ the of NZ a basic of a basic active active site Lys site residue Lys residue that isthat bound is bound to PLP to PLPin step in step1. The 1. Thecarbanion carbanion intermediate intermediate at the at α the-α αcarbon-α carbon is finally is ﬁnally stabilized stabilized by the by expandedthe expanded conjugated conjugated pi-bond pi-bond systems systems with with the theelectron electron sink sink at N-1 at N-1of the of thepyridine pyridine ring ring of PLP. of PLP. 3. β 3.- Elimination:β-Elimination: The The increased increased electron electron density density along along the the conjugated conjugated pi-bond pi-bond is is transferred transferred at at the Cβ-S bond to the proton at the Lys residue, cleaving the C β-S-S bond, bond, which which is is referred referred to to as β-elimination.-elimination. 4. Production Production of of homocysteine: homocysteine: The The product product homocy homocysteinesteine is is released, released, and and the the imine imine is is hydrolyzed. hydrolyzed. The imine is exchange exchangedd with the active site Lys residue. 5. αα-Elimination,-Elimination, production production of of pyruvate pyruvate and and

Crystals 2019, 9, 656 12 of 13

ammonia: The amine at the α-carbon and another pyruvate are released. Simultaneously, the Lys residue is bound to PLP. (b) Cystathionine γ-lyase MccB. 1. Internal aldimine: The Schiﬀ base linkage of PLP to the enzyme Lys residue (internal aldimine) is exchanged with the amine group of the homoserine of cystathionine (external aldimine), which is the opposite side of the amine group of the cysteine. 2. External aldimine: The active site Lys residue abstracts the proton at the Cα of the substrate, which is the same as in step A-2. 3-1. Deprotonation at the β carbon: A base residue in the enzyme active site abstracts a proton at the Cβ of the substrate, and the resulting carbanion is stabilized by the cationic 3 NSB with a new pi-bond between Cβ and Cγ, resulting in sp orbitals at NSB, breaking the expanded conjugated pi-bond system through the pyridine ring of PLP. The base residue is presumed to be Tyr45 interacting with the phosphate group of PLP (Figure4b). 3-2. γ-Elimination: The lone pair electron

of NSB is transferred to the proton at the base residue via the new pi-bond between Cβ-Cγ, cleaving the Cγ-S bond of the substrate. 4, 5. Release of products: The product cysteine is released, and the imine is exchanged to the active site Lys residue with hydrolysis of the imine producing α-ketobutyrate and ammonia. Supplementary Materials: The followings are available online at http://www.mdpi.com/2073-4352/9/12/656/s1, Figure S1: Crystals of SaMccB, Figure S2: SEC-MALS results with purified SaMccB protein, at a flow rate of 0.5 mL/min, and Figure S3: The Superimposition of SaMccB(Yellow) with the structures of XoMetC (green, 0.799 Å RMSD, PDB code: 4IXZ), HpCGS (pink, 0.713 Å RMSD, PDB code: 4L0O), and Human CGL (blue, 0.827 Å RMSD, PDB code: 2NMP). Author Contributions: Conceptualization, D.L., N.-C.H. and A.-R.K.; investigation, D.L., S.J., and J.A.; writing—original draft preparation, N.-C.H.; writing—review and editing, D.L. and A.-R.K.; supervision, N-C.H. and A.-R.K.; funding acquisition, N.-C.H. and A.-R.K. Funding: This research was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry through the Agriculture, Food and Rural Affairs Research Center Support Program, funded by the Ministry of Agriculture, Food and Rural Affairs (710012-03-1-HD120 to NCH). This work was also supported by the National Research Foundation of Korea funded by the Ministry of Education of the Korean government (2017R1D1A1B03033857 to ARK). Acknowledgments: We thank the staff at beamline 5C of Pohang Accelerate Laboratory (Pohang, Korea) for the X-ray diffraction experiments. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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