Crystal Structure of Bacterial Cystathionine -Lyase in The
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crystals Article Crystal Structure of Bacterial Cystathionine G-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 -ketobutyratemethionine,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 sulfurMetB, 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 dicleavageffcontainerent cleavage sites. different While sites. cleavage MetC While MetCexhibitssites. 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 processprocess the γ and-eliminationand can can also also produceprocess produce Hand2 S[H 8can2,S9 ].[8,9] Althoughalso. Althoughproduce molecular Hmolecular2S [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