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Research paper

Genetic and biochemical characterization of Corynebacterium glutamicum ATPphosphoribosyltransferase and its three mutants resistant to feedback inhibitionby histidine

Yun Zhang a, Xiuling Shang a,b, Aihua Deng a, Xin Chai a,b, Shujuan Lai a,b, Guoqiang Zhang a,b,Tingyi Wen a,*aDepartment of Industrial Microbiology and Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, 100101 Beijing, ChinabGraduate University of Chinese Academy of Sciences, 100049 Beijing, China

a r t i c l e i n f o

Article history:Received 22 September 2011Accepted 30 November 2011Available online 8 December 2011

Keywords:ATP phosphoribosyltransferaseCorynebacterium glutamicumFeedback inhibitionHistidineMutagenesis

a b s t r a c t

ATP phosphoribosyltransferase (ATP-PRT) catalyzes the condensation of ATP and PRPP at the first step ofhistidine biosynthesis and is regulated by a feedback inhibition from product histidine. Here, we reportthe genetic and biochemical characterization of such an enzyme, HisGCg, from Corynebacterium gluta-micum, including site-directed mutagenesis of the histidine-binding site for the first time. Genedisruption and complementation experiments showed that HisGCg is essential for histidine biosynthesis.HisGCg activity was noncompetitively inhibited by histidine and the a-amino group of histidine werefound to play an important role for its binding to HisGCg. Homology-based modeling predicted that fourresidues (N215, L231, T235 and A270) in the C-terminal domain of HisGCg may affect the histidineinhibition. Mutating these residues in HisGCg did not cause significant change in the specific activities ofthe enzyme but resulted in the generation of mutant ones resistant to histidine inhibition. Our dataidentified that the mutant N215K/L231F/T235A resists to histidine inhibition the most with 37-foldincrease in Ki value. As expected, overexpressing a hisGCg gene containing N215K/L231F/T235A muta-tions in vivo promoted histidine accumulation to a final concentration of 0.15 � 0.01 mM. Our resultsdemonstrated that the polarity change of electrostatic potential of mutant protein surface preventshistidine from binding to the C-terminal domain of HisGCg, resulting in the release of allosteric inhibition.Considering that these residues were highly conserved in ATP-PRTs from different genera of Gram-positive bacteria the mechanism by histidine inhibition as exhibited in Corynebacterium glutamicumprobably represents a ubiquitously inhibitory mechanism of ATP-PRTs by histidine.

� 2011 Elsevier Masson SAS. All rights reserved.

1. Introduction

Gram-positive actinomycete bacterium Corynebacterium gluta-micum is applied as an industrial workhorse for production ofamino acids and organic acids due to its metabolic and excretingcapabilities [1e3]. As its genome sequence became available andmany metabolic regulatory mechanisms have been extensively

elucidated, C. glutamicum now serves as a model bacterium forconstruction of engineered strains on the base of a rational schemeincluding a series of genetic modifications [4e6]. Given that carbonflux through the pentose phosphate pathway (PPP) is higher thanthat through the TCA cycle [7,8], C. glutamicum is, therefore, anappropriate candidate for metabolic engineering of histidinebiosynthesis with its precursor supplied through PPP pathway. Todate, de novo engineering of histidine biosynthesis in C. glutamicumis not reported yet due to lack of knowledge about the functionalrole of its key enzyme and the regulation mechanism of histidinebiosynthesis in vivo.

ATP phosphoribosyltransferase (ATP-PRT, EC 2.4.2.17) is the firstenzyme in histidine biosynthesis pathway and plays a criticalregulatory role in controlling carbon flux towards histidine biosyn-thesis [9]. In the presence of divalent magnesium ion, ATP-PRTcatalyzes the condensation of 5-phosphoribosyl 1-pyrophosphate(PRPP) and ATP to form N1-(5-phosphoribosyl)-ATP (PR-ATP).

Abbreviations: HisGCg, ATP phosphoribosyltransferase from Corynebacteriumglutamicum; ATP-PRT, ATP phosphoribosyltransferase; CD, circular dichroism; DTT,dithiothreitol; kDa, kilo Dalton; bp, base pairs; SDS-PAGE, soduim dodecyl sulphatepolyacrylamide gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; PCR, polymerase chain reaction; PDB, protein data bank;PPP, pentose phosphate pathway; PRPP, 5-phosphoribosyl 1-pyrophosphate;PR-ATP, N1-(5-phosphoribosyl)-ATP; N (or Asn), asparagine; K (or Lys), lysine; L (orLeu), leucine; F (or Phe), phenylalanine; T (or Thr), threonine; A (or Ala), alanine.* Corresponding author. Tel.: þ86 10 62526173; fax: þ86 10 62522397.

E-mail address: [email protected] (T. Wen).

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Biochimie

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0300-9084/$ e see front matter � 2011 Elsevier Masson SAS. All rights reserved.doi:10.1016/j.biochi.2011.11.015

Biochimie 94 (2012) 829e838


The best studied ATP-PRT is that fromGram-negative Escherichia coliand Salmonella typhimurium [9e12]. The reaction catalyzed by ATP-PRT is reversible and both directions are consistent with a Bi-Bikinetic mechanism [10]. AMP and ADP are competitive inhibitorswith respect to both substrates and become positively cooperativeinhibitors in the presence of histidine [11,12]. ATP-PRT activity issubjected to feedback inhibitionby the level of intracellularhistidineto achieve strict regulation of histidine biosynthesis [9].

ATP-PRT is a member of a fourth (type IV) phosphoribosyl-transferase (PRT) subfamily of enzymes, which are essentiallyinvolved in many pathways for the biosynthesis of cofactors, aminoacids and nucleotides as well as the salvage of nucleotides [13]. Asencoded by the hisG gene, ATP-PRTs have been found in archaea,bacteria, fungi and plants and share low sequence identity amongvarious species [9,14,15]. These enzymes comprise two structurallydistinct subfamilies referredasHisGL or long formandHisGS or shortform, respectively. HisGL is composed of about 280e310 amino acidsand found in lower eukaryotes and bacteria such as E. coli,S. typhimurium andMycobacterium tuberculosis. In contrast, HisGS isabout 80 residues shorter than HisGL at its C terminus and has nocatalytic activity unless they bind the regulatory subunit HisZ as inLactococcus lactis and Thermotoga maritima [16]. Crystal structuresof ATP-PRTs have provided clues to understand the catalytic andregulatory mechanism of two subfamily enzymes. Enzymes fromthe short form ATP-PRT subfamily are hetero-octamers andcomposed of four catalytic HisGS subunits and four HisZ subunits.The latter acts as a regulatory domain to compensate for the absenceof a C-terminal regulatory domain found in HisGL. Therefore, HisGSexhibits the same catalytic mechanism as HisGL but with a differentregulatory pattern [17e19]. To date, two different structures ofhetero-octameric ATP-PRT have been identified to exist as a histi-dine-inhibited complex in T. maritima [18] and ATP-activated andPRPP-bound forms in L. lactis [17,19]. Enzymes from the HisGLsubfamily are homo-hexamers which are the inactive, histidine-binding forms of the enzyme. The active form of HisGL is a homo-dimer and each subunit is composed of three distinct domains [20].There are twodifferentHisGL structures. In E. coli, the PRPPandpartsof the ATP-binding sites locate in the first two domains in ATP-PRTstructure, which clearly identifies that AMP can bind in bothPRPP- and ATP-binding sites [21]. In M. tuberculosis, a comparisonbetween the apo and AMP:His forms of ATP-PRT structure hasrevealed that the C-terminal domain directly interacts with histi-dine. This binding triggers the hexamer conformation change andthus leads to the steric hindrance in the active site [22]. BothofHisGLstructures exhibit similar allosteric inhibition by histidine despitethe fact that large differences exist in their amino acid sequences.However, there is no further study performed to identify or analyzethe histidine binding sites in HisGL.

The hisGL gene (GenBank accession no. AF050166) had beencloned previously from a genome library of C. glutamicum strainASO19 by complementation of E. coli histidine auxotroph [23].Genome sequencingofC. glutamicum strainATCC13032 showed thatthis strain contains a gene called hisGCg (NCgl1447) that encodesa protein annotated as anATP-phosphoribosyltransferase [24,25]. Inthis study, we identified that hisGCg gene encodes a functionalATP-PRT essential for histidine biosynthesis in C. glutamicum andshowed detailed biochemical characterization performed on a longform ATP-PRT from a Gram-positive bacterium. According tosequence alignment and homology-based modeling using thecrystal structure of ATP-PRT of M. tuberculosis [22], four residues inthe C-terminal domain of HisGCg were predicted to be involved inhistidine binding. Functional analysis of HisGCg mutants confirmedthat three of the four residues were crucial to release the feedbackinhibition by histidine. The findings about binding between histi-dine and HisGCg as well as the electrostatic potential of protein

surface account for the resistance of HisGCg to histidine, sheddinglight on the molecular basis of how long-form ATP-PRTs areinhibited by histidine feedback.

2. Materials and methods

2.1. Bacterial strains, plasmids and growth media

E. coliDH5a (F־־ supE44F80 dlacZ ΔM15 Δ(lacZYA-argF)U169 endA1recA1 hsdR17 (rk

- ,mkþ) deoR thi-1 l־־ gyrA96 relA1) and BL21(DE3) (hsdSgal (lcIts857 ind-l Sam7 nin-5 lacUV5-T7 gene 1)) were used in thiswork as host strains for the gene cloning and expression of therecombinant enzymes, respectively. Plasmid pET28a (Novagen) wasused forproteinexpression.E. coli strainswere culturedaerobicallyona rotary shaker at 180 rpm in Luria-Bertani (LB) medium supple-mentedwith50mg/mLofkanamycinwhenneeded.TheC. glutamicumRES167 (restriction-deficient mutant of ATCC13032, DcglIMDc-glIRDcglIIR) was used for genetic disruption and complementationusing plasmids pK18mobsacB and pXMJ19 [26,27]. C. glutamicumstrains were aerobically grown at 30 �C on a rotary shaker at 200 rpmin richmediumCGIII [28] or in theminimalmediumCGX [29]. For thegeneration of mutant and maintenance of C. glutamicum brain heartbroth with 0.5 M sorbitol mediumwas used [30].

2.2. Genetic disruption and complementation in C. glutamicum

Disruption of hisGCg was performed by replacing its 30-endsequence (corresponding to nucleotide 583e846) with the chlor-amphenicol resistance cassette which was amplified using pXMJ19as template [27]. Three pairs of primers listed in SupplementalTable 1 were used to amplify the hisGCg gene (163e582 bp),chloramphenicol resistance gene (861 bp) and downstreamsequence of hisGCg (408 bp). The products were fused by the spliceoverlap extension (SOE) PCR and ligated into pK18mobsacB toconstruct plasmid pK18mobsacBDhisGCg. The resulting mutantRES167DNCgl1447 with disruption of hisGCg gene in C. glutamicumRES167 was constructed by integration and excision of the plasmidpK18mobsacBDhisGCg according to the method of Schäfer et al [26].

For complementation, the plasmid pXMJ19-hisGCg containingan intact hisGCg gene was introduced into the C. glutamicumRES167DNCgl1447 and the expression of hisGCg was induced byisopropylthio-b-D-galactopyranoside (IPTG).

2.3. Expression and purification of wild type HisGCg and its mutants

The mutated hisGCg genes were constructed by SOE PCR usinga series of mutagenic primers as shown in Supplemental Table 1.Mutant andwild type geneswere cloned into the vector pET28a andexpressed in the BL21 (DE3) strains. The preparation of plasmid,digestion with restriction endonucleases, and electroporation werecarried out as described by Sambrook et al. [31]. Cells were inducedwith 1 mM IPTG for 8 h at 16 �C when the OD600 reached 0.6, andthen harvested by centrifugation and resuspended in the standardbuffer (10 mM TriseHCl, pH 7.5, 100 mM NaCl, 0.5 mM EDTA and1mMDTT). The suspended cells were disrupted by sonication in anice-water bath and the supernatants were collected by centrifuga-tion at 12,000� g for 20min at 4 �C. The N-terminal 6�His-taggedHisGCg proteins were purified by His-Bind column chromatographyaccording to the manufacturer’s protocol (Novagen). The elutedprotein solution was desalted in HiTrap columns (GE Healthcare,USA) and concentrated by ultrafiltration (10-kDa MW cut-offmembrane, Millipore, USA). Subsequently, the HisGCg was appliedto a Superdex 200 10/300 GL column (10 � 300 nm, GE Healthcare,USA) equilibrated with the standard buffer using the ÄKTA purifiersystem (GEHealthcare, USA) and eluted at a flow rate of 0.5mL/min.

Y. Zhang et al. / Biochimie 94 (2012) 829e838830


The fractions containing wild type or mutant HisGCg were pooledand concentrated for further analysis as described above. Puritywasjudged after SDS-PAGE and staining with Coomassie Brilliant Blue.Protein concentrations were determined by measuring the absor-bance at 595 nm and by the Bradford method using the Bio-RadProtein Assay Reagent (Bio-Rad, USA) with bovine serum albumin(BSA) as the standard.

2.4. Molecular weight determination

Matrix-assisted laser desorption/ionization time of flight(MALDI-TOF) mass spectra were obtained on an Ultraflex MALDI-TOF MS (Bruker, Daltonik, Bremen, Germany) in the positivereflection mode with the laser energy of 84 Hz. Mass spectra werecalibrated using the protein calibration standard as external stan-dards. The native molecular mass of HisGCg was detected by gelfiltration chromatography on a Superdex 200 10/300 GL column(GE Healthcare, USA), equilibrated with 0.05 M potassium phos-phate buffer (pH 7.2) containing 0.15 M NaCl and 1 mM DTT.

2.5. Circular dichroism spectroscopy

Far-UV Circular Dicroism (CD) was recorded on a JASCO J-810spectropolarimeter (Jasco, Japan). The purified HisGCg or the triplemutant protein was dissolved in 20 mM sodium phosphate buffer(pH 7.5). Spectral analysis was taken over a wavelength range from190 to 260 nm in cuvettes with a 2-mm path length at 0.5-nmintervals under constant nitrogen flush and the bandwidth of1 nm. All spectra were recorded at room temperature, and threescans were averaged with the blank subtracted.

2.6. Enzymatic activity and property determination

The assay for ATP-PRT activity was based on the high extinctioncoefficient of PR-ATP at 290 nm [32]. The reaction mixture con-tained 100mM TriseHCl (pH 8.5), 150mMKCl, 10mMMgCl2, 5 mMATP, 0.5 mM PRPP, 1 U yeast pyrophosphatase, and the enzyme ina total volume of 0.5 mL. Reactions were carried out in quartzcuvettes with a path length of 1 cm. The increase in the absorbanceat 290 nm was monitored by a UVevisible spectrophotometer(2802 UV/VIS, Unico, Shanghai, China) at room temperature for4 min, reaction mixtures without enzyme were used as blankcontrol. One unit of activity was defined as the amount of enzymecapable of increasing the absorbance by 0.02 per min, corre-sponding to the transformation of 1.67 nmol of the substrate permin [33].

The effect of temperature on HisGCg activity was assayed in thestandard reaction condition at a temperature range of 20e50 �C.The reactionmixture was pre-incubated at the desired temperaturefor 10 min and the assay was started by adding enzyme. To deter-mine the pH effect on the enzyme activity, the following bufferswere used at 100 mM: sodium phosphate buffer (pH 6.0, 6.5, 7.0and 7.5), TriseHCl buffer (pH 8.0, 8.5 and 9.0) and glycine-NaOHbuffer (pH 9.5, 10.0, 11.0, 12.0 and 13.0). CoCl2, NiSO4, CaCl2,MnSO4, ZnCl2, HgSO4, CuSO4, FeSO4, Fe2(SO4)3, AgNO3, EDTA andH2O2 were applied to determine the effects of various metal ionsand chemicals on HisGCg under the assay condition. The enzymaticactivity in the absence of chemicals was set at 100%. The data wereobtained from three independent experiments.

To evaluate the effect of histidine on the enzymatic activity,histidine were added to the enzyme assay mixture containingdifferent buffers at the pH range from 7.5 to 11.0. The reactionwithout histidine was run in parallel and the activity in the absenceof histidine was set at 100%. The data were obtained from threeindependent experiments.

2.7. Kinetic measurements

To determine the kinetic mechanism of HisGCg, the reactiontypically consisted of 100mMTriseHCl (pH8.5),150mMKCl,10mMMgCl2, 1 U yeast pyrophosphatase and variable concentrations ofATP and PRPP. The PRPP concentration varied from 0.04 to 0.25 mMat saturated concentration of 5 mM ATP or the ATP concentrationvaried from 0.1 to 2.0 mM at saturated concentration of 0.5 mMPRPP. We confirmed that the product formation was linearthroughout this period. Steady-state parameters were determinedby fitting the curve to v ¼ Vmax [S]/(Km þ [S]), where v is the initialvelocity and S is the variable substrate concentration. The kcat valuewas calculated basedonVmax and themolecularweight of 32 kDa forthe subunit. The inhibition constant (Ki) value of AMP and ADPversus ATP were determined by Dixon plot [34]. The inhibitionconstant of the HisGCg by histidine was determined from velocityversusPRPP concentrationplots at afixedATP concentration (5mM),and over histidine concentrations ranging from 0 to 600 mM. Allkinetic parameters were obtained from at least five measurements.

2.8. Sequence analysis and homology modeling

The genomic sequence of C. glutamicum ATCC13032 (GenBankaccession no. NC_003450) was retrieved from GenBank. Sequencecomparisons and database searches were carried out using BLASTprograms at the BLAST server of National Center for BiotechnologyInformation website (http://www.ncbi.nlm.nih.gov). Pairwise andmultiple protein sequence alignments were made with the CLUS-TAL X program [35]. The alignment of secondary structure wasproduced using ESPript 2.2 online (http://espript.ibcp.fr/ESPript/ESPript/), based on a knowledge of the high resolution x-ray crys-tallographic structure ofM. tuberculosis (PDB code 1NH7) and E. coliATP-PRTs (PDB code 1H3D) [36]. The automated comparativeprotein structure homology-modeling server, SWISS-MODEL(http://swissmodel.expasy.org) was used to generate the three-dimensional model of active and inhibited HisGCg based on thecrystal structure of ATP-PRT from M. tuberculosis (PDB code 1NH7and 1NH8) [37]. The Deep View Swiss-PDB Viewer software fromthe EXPASY server (http://www.expasy.org/spdbv) and PyMOL v1.0

Fig. 1. Growth curves of C. glutamicum RES167 wild type and RES167DNCgl1447mutant cells. Cells were grown in the minimal medium CGX at 30 �C, rotatory shakingof 200 rpm for 24 h. Data are average of three parallel cultures. C, RES167; :,RES167DNCgl1447; -, RES167DNCgl1447 supplemented with NCgl1447.

Y. Zhang et al. / Biochimie 94 (2012) 829e838 831


(http://www.pymol.org) were used to visualize and analyze thethree-dimensional model.

3. Results

3.1. C. glutamicum hisGCg gene is essential for histidine biosynthesis

C. glutamicum genome contains a hisGCg (NCgl1447) gene thatencodes a protein annotated as a putative ATP phosphoribosyl-transferase (HisGCg) [24,25]. HisGCg was predicted to be a long form

ATP-PRT composed of 281 amino acid residues with a theoreticalmolecular mass of 31 kDa. Sequence analysis revealed that HisGCgshares moderate sequence identity with characterized ATP-PRTfrom M. tuberculosis (65%) and predicted ATP-PRTs from Rhodo-coccus equi (67%) and Nocardia farcinica (63%), but low sequence

Fig. 2. Purification and identification of recombinant HisGCg in vitro. (A) SDS-PAGE ofprotein fractions at different stages of HisGCg and mutants purification. SDS-PAGE wasperformed with 5% stacking gel and 12% resolving gel. The gels were visualized withCoomassie blue R-250. Lane 1, supernatant of crude extract from E. coli BL21(DE3)/pET28a; Lane 2, supernatant of crude extract from E. coli BL21(DE3)/pET28a-hisGCg,Lane 3, HisGCg after His-tag affinity; Lane 4, HisGCg after gel filtration chromatography;Lane 5, The mutant N215K after gel filtration chromatography; Lane 6, The mutantL231F/T235A after gel filtration chromatography; Lane 7, The mutant A270P after gelfiltration chromatography; Lane 8, The mutant N215K/L231F/T235A after gel filtrationchromatography; Lane 9, The mutant N215K/L231F/T235A/A270P after gel filtrationchromatography. (B) MALDI-TOF mass spectrum of purified recombinant HisGCg. Themolecular mass of HisGCg monomer was determined to be 32191.15 kDa. (C) molecularmass determination by gel filtration chromatography. HisGCg was represented asa triangle (:). The standard proteins were represented by circles (C): 1, Ovalbumin(44 kDa); 2, Conalbumin (75 kDa); 3, Aldolase (158 kDa); 4, Ferritin (440 kDa); 5,Thyroglobulin (669 kDa). Kav¼ (Ve-Vo)/(Vc-Vo), with Ve the elution volume of theprotein, Vo the void volume of the column, and Vc the total bed volume of the column.

Fig. 3. Effects of temperature, pH, various metal ions and chemicals on activity ofrecombinant HisGCg. (A) The recombinant HisGCg was incubated in 100 mM TriseHClbuffer (pH 8.5) at various temperatures (20-50 �C). (B) The recombinant HisGCg wasincubated at 25 �C in various pHs. 100 mM sodium phosphate buffer (pH 6.0e7.5),100 mM TriseHCl buffer (pH 8.0e9.0) and 100 mM glycine-NaOH buffer (pH 9.5e13.0).(C) The recombinant HisGCg was incubated in 100 mM TriseHCl buffer (pH 8.5) con-taining various metal ions and chemicals.



identity with bacterial hexameric ATP-PRT from E. coli (36%) andhetero-octameric ATP-PRTs from L. lactis (30%) and T. maritima(20%) and eukaryotic ATP-PRT from yeast Saccharomyces cerevisiae(34%). To test the function of HisGCg in vivo, a hisGCg-null C. gluta-micum mutant, RES167DNCgl1447, was created with the 30-end297-bp sequence of hisGCg gene substituted by a chloramphenicolresistant gene. Disruption of hisGCg gene in RES167DNCgl1447 wasconfirmed by PCR amplification and direct nucleotide sequencingof the PCR products. Growth test showed that RES167DNCgl1447fails to grow on minimal medium (Fig. 1) and its growth could berestored when the minimal medium was supplemented withhistidine (data not shown). Furthermore, complementation ofRES167DNCgl1447 with wild type hisGCg gene restored its growthwhen cultured in minimal medium (Fig. 1). Taking the conclusiveevidence all together, hisGCg is an essential gene for histidinebiosynthesis in C. glutamicum.

3.2. Purification and identification of the recombinant HisGCgin vitro

Very little is known about the function of a long form ATP-PRTfrom Gram-positive bacteria so far, but HisGCg provided us a greatopportunity to investigate the role of such an enzyme in histidinebiosynthesis. To functionally characterize this protein, we firstsought to generate an N-terminal 6 � His-tagged recombinantHisGCg (His6-HisGCg) in E. coli BL21 (DE3). After purification by His-tag affinity and gel filtration chromatography (Fig. 2A) therecombinant His6-HisGCg showed an approximately 13.6-foldincrease in purity with a recovery of 22.2% and a specific activityof 2356.4 U/mg (Supplemental Table 2). The subunit molecularweight of HisGCg determined by MALDI-TOF MS was 32191 Da(Fig. 2B). Gel filtration of the recombinant HisGCg yielded twofractions with estimated molecular masses of 192 kDa and 63 kDa,corresponding to the predicted sizes of a homohexamer anda homodimer (Fig. 2C).

3.3. Enzymatic properties of the recombinant HisGCg

HisGCg was active at temperatures between 20 �C and 50 �Cwitha maximal activity achieved at 30 �C (Fig. 3A), the optimaltemperature for S. typhimurium ATP-PRT [33]. HisGCg maintainedover 40% relative activities in pH range between 7.5 and 13.0 andrapidly lost its activity when pH dropped below pH 7.5 (Fig. 3B). Themaximal activity of HisGCg was observed at pH 10.0, higher than theoptimal pH of S. typhimurium ATP-PRT [32]. Effects of various metalions and chemicals on the activity of HisGCg were also determinedas well. As shown in Fig. 3C, Co2þ hardly affected HisGCg activity, butCa2þ, Mn2þ, Zn2þ and Hg2þ showedmoderate inhibitory effect witha residual activity about 40% at 1 mM concentration. Cu2þ, Fe3þ,Fe2þ and Agþ inhibited HisGCg activity completely, which wasincongruent with what was observed for the ATP-PRT inS. typhimurium [38]. Interestingly, Ni2þ was the only metal ion that

shows a stimulatory effect on HisGCg activity. Different from whatwas observed for S. typhimurium that the addition of Ni2þ or Hg2þ

desensitizes ATP-PRT from histidine inhibition [38], histidinesignificantly inhibited HisGCg activity in the presence of Ni2þ orHg2þ (data not shown). In addition, the chelator EDTA reducedHisGCg activity by 37% at 1 mM and by 71% at 5 mM. Addition of theoxidizing agent hydrogen peroxide (1 mM) suppressed HisGCgactivity by 73%.

Table 1Kinetic parameters of ATP-PRTs from different species.

Organism Km(PRPP) (mM) Km(ATP) (mM) kcat(PRPP) (s�1) Ki(His) (mM) Ki(AMP) (mM) Ki(ADP) (mM) Reference

C. glutamicum 0.08 � 0.01 0.22 � 0.02 1.91 � 0.14 0.11 � 0.02 1.29 � 0.42 0.88 � 0.35 This studyE. coli 0.11 0.8 NDa ND ND ND [39]S. typhimurium 0.067 0.2 ND 0.1 5 7 [38]S. typhimurium 0.011 0.11 2.7 0.07 5 ND [10]L. lactis 0.018 � 0.003 2.7 � 0.26 2.67 � 0.27 0.081 1.44 ND [40]A. thaliana ATP-PRT1 0.13 0.60 ND 0.045b ND ND [41]ATP-PRT2 0.57 0.51 ND 0.32b ND ND [41]

a ND, Not determined.b The parameter values of histidine inhibition for two A. thaliana ATP-PRTs are reported as IC50.

Fig. 4. The feedback inhibition effects by histidine on recombinant HisGCg underdifferent pH. (A) The recombinant HisGCg was incubated with 0.5 mM histidine indifferent buffers at the pH range from 7.5 to 11.0. (B) The recombinant HisGCg wasincubated with histidine (0.01e2 mM) in pH 7.5 (-), pH 8.5 (C), pH 9.5 (:) and pH11.0 (A) buffers, respectively.



Fig. 5. Model of the 3-D structure of HisGCg and secondary structural alignment of ATP-PRTs. (A) model of the HisGCg monomer structure. Homology model generated with SWISS-MODEL based on the 3D-structure of ATP-PRT from M. tuberculosis (PDB code 1NH7). Ribbon representation of the monomeric HisGCg subunit colored according to secondarystructure. The N and C termini of the HisGCg polypeptide chain and the secondary structural elements are labelled. The residues involving in the PRPP (a) and histidine (b) molecule



3.4. Kinetic parameters of the recombinant HisGCg

Kinetic analyses showed that HisGCg had a higher affinity forPRPP than ATP (Table 1 and Supplemental Fig. 1). The Km value ofHisGCg for PRPP (0.08 � 0.01 mM) was comparable to that of otherlong form ATP-PRTs but 4-fold higher than that of the short formenzyme from L. lactis [39e41], despite the same conserved residuesfor the PRPP binding in two subfamily ATP-PRTs. The kcat value(1.91 � 0.14 s�1) of PRPP was similar to those of other ATP-PRTs,indicating no obvious difference of two form ATP-PRTs in thecatalytic activity. AMP and ADP were competitive inhibitors to ATPwith the Ki values of 1.29 � 0.42 and 0.88 � 0.35 mM, respectively,which both were apparently lower than those determined fromother ATP-PRTs (Table 1). Histidine was noncompetitive inhibitorwith the Ki value of 0.11 � 0.02 mM for PRPP, higher than those ofATP-PRTs from S. typhimurium (0.07e0.1 mM) [10,38] and L. lactis(0.081 mM) [40] but lower than that of ATP-PRT from T. maritima(0.35 � 0.02 mM) [18], indicating the inhibitory effects by histidineare different on various ATP-PRTs. In addition, the CD spectra ofHisGCg showed little difference in the absence or presence of 5 mMhistidine (Supplemental Fig. 2A), which was consistent with thatobserved for ATP-PRT from E. coli [42], indicating that the binding ofhistidine does not lead to the transformation of the secondarystructure of HisGCg.

3.5. Alkaline pH decreases the inhibitory effect of histidine

The inhibitory effect of histidine on ATP-PRTactivity is histidine-specific since intermediates in histidine biosynthesis pathway andstructural analogues of histidine do not cause ATP-PRT inhibition[38]. As assayed at different pH conditions, histidine varied signif-icantly in its inhibitory effect on HisGCg activity. In the presence of0.5mMhistidine, the activity of HisGCg was attenuated to 12% of thecontrol without histidine at pH 7.5 and to 88% of the control at pH11.0 (Fig. 4A). When the histidine concentration was increased to2 mM, the relative activity of HisGCg was 3% at pH 7.5 and 50e60%at pH 9.5 or pH 11.0 (Fig. 4B). In terms of the pKa value of histidine,the charge state of histidinewas converted from neutral to negativewhen the pH value shifted from 7.5 to 11.0 due to deprotonation ofthe a-amino group. The decrease in inhibitory effect of histidinesuggested that the charge state of a-amino group of histidine atalkaline pH affects its binding efficiency. These data indicated thatthe a-amino group of histidine is critical in determining its bindingto HisGCg.

3.6. Homology modeling of HisGCg and analysis of its sites forhistidine binding

By using SWISS-MODEL server and ESPript program, here wepresented a structure model of HisGCg based on the crystal struc-ture ofM. tuberculosis ATP-PRT (1NH7.pdb) [22] and the similaritiesbetween the predicted secondary structure of ATP-PRTs frombacterium, archaea and yeast (Fig. 5A, B). According to this model,HisGCg is an L-shaped monomer and composed of nine a-helixesand fifteen b-sheets that form three distinct domains (Fig. 5A). Thismodel also showed that residues involved in PRPP binding were

conserved in both hexameric and hetero-octameric ATP-PRTs(Fig. 5B), indicating that the two subfamilies of ATP-PRT evolvedfrom the same origin.

Previous kinetics analysis showed that at least two interactiveATP-PRT sites are responsible for histidine binding [43], whereascrystal structures of ATP-PRTs from E. coli and M. tuberculosisshowed that several different residues of these proteins areinvolved in histidine binding [21,22]. According to the structuremodel of M. tuberculosis ATP-PRT, Asp218 and Thr238 (His232 andThr252 in E. coli ATP-PRT) participates in binding to the aminogroup of histidine and Ala273 (Ser288 in E. coli ATP-PRT) formsa hydrogen bond with the R-group of histidine [21,22]. Other thanthese, it was noticed that Leu234 and Leu253 contact directly withhistidine to form a hydrogen bonding network [22]. Similar toM. tuberculosis ATP-PRT, our model revealed that Asn215, Leu231,Thr235 and Ala270 locate in the C-terminal domain of HisGCg andare very close to the histidine binding site (Fig. 5C). Sequencealignment revealed that Asp215, Leu231, Thr235 and Ala270 arehighly conserved among ATP-PRTs from different genera of Gram-positive bacteria except that the Asp is replaced by an Asn inATP-PRTs from Corynebacterium genus (Supplemental Fig. 3).

3.7. Kinetic analysis of the HisGCg mutants

To investigate the role of the four conserved residues (Asn215,Leu231, Thr235 and Ala270) of HisGCg in histidine binding, HisGCgmutants including N215K, A270P, L231F/T235A, N215K/L231F/T235Aand N215K/L231F/T235A/A270P were generated by site-directedmutagenesis. In these mutants, the polar side chain was replacedwith a cationic or nonpolar sidechain, while steric factors wereaddressed by replacing the residue with other hydrophobic aminoacid of a different size. All five mutants can be readily expressed andpurified to apparent homogeneity, suggesting that themutations didnot affect protein stability (Fig. 2A). Themutantswere similar towildtype HisGCg regarding to their specific activitywith an exception thatthemutant N215K/L231F/T235A/A270P exhibited a 5.6-fold decrease(Table 2). Mutants N215K, A270P, L231F/T235A and N215K/L231F/T235A were resistant to histidine inhibition to different extents,demonstrating that histidine binds HisGCg via these four amino acids(Fig. 6). As determined in the presence of 2mMhistidine, themutantN215Kpreserved 51% specific activitywhichwas 4.8-fold higher thanthat of wild type enzyme. For the mutant A270P, only 36% specificactivity as compared to wild type protein remained, revealing thatA270 residue plays a minor role in histidine binding. The mutantLeu231/Thr235 retained 60% specific activity, whichwas 1.2- and 1.7-folds higher than that of mutants N215K or A270P, indicating thatthese two residues are important in stabilizing histidine binding. Thetriple mutant N215K/L231F/T235A was much less sensitive to histi-dine inhibition. Even in the presence of 4 mM histidine, this mutantstill maintained 60% specific activity, which was 13- and 2-foldshigher than that of wild type HisGCg and the single mutants,respectively. CD spectra analysis did not find major differencesbetweenwild typeHisGCg and the triplemutantN215K/L231F/T235A,indicating that amino acid substitutions hardly cause significantdisruption in global secondary structure of HisGCg (SupplementalFig. 2B).

binding are shown in ball representation colored by type of atom. (B) structure-based multiple alignment of the amino acid sequences of ATP-PRTs from bacterium, archaea andyeast, produced using ESPript 2.2. The a-helices, p-helices and b-sheets are represented as helices and arrows, respectively, and strict b-turns are marked with TT letters. Theresidues involved in binding PRPP are marked - in blue, and four residues shown to be involved in histidine binding with a pink +.This sequence alignment was created using thefollowing sequences: Mt_HisG (M. tuberculosis, GenBank No.O33256), Cg_HisG (Corynebacterium glutamicum ATCC13032, GenBank No.AAD02497), Sc_HisG (Saccharomycescerevisiae, GenBank No.P00498), St_HisG (Salmonella typhomurium, GenBank No.P00499), Ec_HisG (Escherichia coli GenBank No.P10366), Bs_HisG (Bacillus subtilis GenBankNo.O34520), La_HisG (Lactococcus lactis, GenBank No.Q02129), Tm_HisG (Thermotoga maritima, GenBank No.Q9X0D2). (C) Superimposition of HisGCg structure with/withouthistidine binding. The active and inhibited structures are shown in blue and green ribbon model, respectively. The histidine is shown in ball representation colored by type of atom.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)



The four mutants had the significant increases in Ki value forhistidine compared to wild type HisGCg (Table 2 and SupplementalFig. 4). The mutant L231F/T235A exhibited a 4.7-fold increase in Kivalue thanwild type HisGCg. The triple mutant N215K/L231F/T235Aresisted to histidine inhibition the most with 37- and 8-foldincreases in Ki value than wild type HisGCg and the mutant L231F/T235A, respectively. Moreover, the mutant L231F/T235A exhibiteda slight increase in Km values for PRPP and ATP, whereas othermutants were much less affected at the level of Km, suggesting thatthe mutations of the histdine-binding residues have no effect onthe affinity of PRPP and ATP to the enzyme (Table 2). Nevertheless,the mutants except for N215K exhibited the decreased values forkcat, indicating that the mutations of L231F, T235A and A270P affectthe catalytic activity of HisGCg (Table 2).

3.8. Overexpression of HisGCg containing N215K/L231F/T235Amutations promotes the histidine accumulation in vivo

To evaluate the effect of histidine feedback-resistant HisGCgmutants in vivo, shake-flask fermentation was carried out usingC. glutamicum ATCC13032 strains overexpressing HisGCg or thetriple mutant N215K/L231F/T235A in minimal medium. No histi-dine could be detected in the hisGCg-expressing strain. In contrast,0.15 � 0.01 mM histidine was accumulated when the feedbackinhibition of HisGCg was released as shown in the triplemutant case(Supplemental Fig. 5).

4. Discussion

Many key enzymes involved in amino acid biosynthesis path-ways suffer the feedback inhibition by metabolic end-products.

This makes them important regulating switches in controlling themetabolic flux towards the amino acids biosynthesis pathway. Asfor histidine biosynthesis pathway, its first enzyme, ATP-PRT, issubjected to the inhibitory regulation by product histidine [38]. Thelong-form ATP-PRT exists in equilibrium between its active dimericform and inactive hexameric form [44]. PR-ATP, AMP and histidinepromote the formation of hexameric ATP-PRT, whereas PRPP andlow enzyme concentrations seem to cause the dissociation ofhexamers into dimers [45]. Similar to a previous report [46], HisGCgexists in different oligomeric states and the inhibitory effect ofhistidine on HisGCg activity was significantly improved whenhistidine concentration increased. The structural similarity ofHisGCg to other long-form ATP-PRTs makes it likely that its inhibi-tion by histidine likewise involves the transition between theobserved dimeric and hexameric forms, which are the putativeactive and inactive states.

Two subfamilies of ATP-PRTs show significantly different modesfor histidine binding. For long form ATP-PRTs, one histidine mole-cule interacts with each subunit of a hexameric ATP-PRT within itsC-terminal domain, which results in a conformation change tostabilize inactive hexamer [21,22]. For short form ATP-PRTs, twohistidine-binding sites are located at each of four HisG/HisZ inter-faces in a hetero-octameric complex [18]. Our results showedthat four residues (Asn215, Leu231, Thr235 and Ala270) in theC-terminal domain of HisGCg are targeted sites for histidinebinding. Although mutation of a single residue has a marginalcontribution in releasing histidine inhibition mutating multi-residues simultaneously can significantly increase the resistanceof HisGCg to histidine inhibition. Based on the structure comparison(Fig. 7A), we conclude that the histidine inhibition against HisGCg isreleased if the following factors are met. First, the change froma polar side chain to a positive side chain at position 215 increasesa strong electrostatic repulsive effect to the a-amino group ofhistidine. Second, the aromatic ring at position 231 increases sterichindrances of histidine binding. Besides, the deficiency of hydroxylgroups at position 235 makes it impossible to interact with the a-amino group of histidine by hydrogen bonds. Consequently, weinfer that the binding between histidine and HisGCg is stabilized byseveral reciprocal forces and the hydrogen bonds formed with thea-amino group of histidine play a major role in this event.

Considering that the charge state of the a-amino group inhistidine was essential to bind to HisGCg, we further analyzed theelectrostatic potential of protein surface using the package APBS(Fig. 7B). In wild type protein, the surface in C-terminal residuesregion which was responsible for binding histidine exhibiteddistinct negative electrostatic potential. This benefited the inter-actionwith the positive charge of a-amino group in histidine by theelectrostatic force and stabilized the inhibited conformation.However, the polarity of C-terminal domain of the mutant N215K/L231F/T235A changed from negative to positive. The positiveelectrostatic potential increased the electrostatic repulsive force toprevent histidine from binding to the C-terminal domain of HisGCg.This did not result in the allosteric inhibition, thus shedding light

Table 2Kinetic parameters for wild type HisGCg and its mutants.

ATP-PRT Specific activity(mmol/min/mg)

Km(PRPP) (mM) Km(ATP) (mM) kcat(PRPP) (s�1) Ki(His) (mM)

Wild type 2.19 � 0.09 0.08 � 0.01 0.22 � 0.02 1.91 � 0.14 0.11 � 0.02N215K 2.01 � 0.14 0.08 � 0.02 0.22 � 0.03 2.22 � 0.02 0.28 � 0.01A270P 1.96 � 0.14 0.07 � 0.01 0.24 � 0.05 1.29 � 0.07 0.24 � 0.03L231F/T235A 2.04 � 0.15 0.09 � 0.02 0.35 � 0.03 1.36 � 0.02 0.52 � 0.02N215K/L231F/T235A 2.10 � 0.12 0.08 � 0.01 0.23 � 0.04 1.75 � 0.06 4.15 � 0.21N215K/L231F/T235A/A270P 0.39 � 0.11 NDa ND ND NDa ND, Not determined.

Fig. 6. The specific activities of HisGCg and its mutants in the presence of differentconcentrations of histidine. The bars represent means of at least three experiments andthe standard deviation was marked. *, p < 0.05 and #, p < 0.01 compared with the wildtype HisGCg at the same concentration of histidine.



on the feedback inhibition mechanism of long form ATP-PRTs.Furthermore, sequence identity comparison made among HisGCgand ATP-PRTs from different genera of Gram-positive bacteriademonstrated that four residues are highly conserved in the hex-amer ATP-PRTs, indicating that this is a ubiquitously inhibitorymechanism by histidine.

Results from shake-flask fermentation showed that over-expression of the triple mutant HisGCg in vivo promoted thehistidine accumulation, thus demonstrating that the release offeedback inhibition by histidine contributes to the increase of themetabolic flux towards histidine biosynthesis. Therefore, themutant N215K/L231F/T235A of HisGCg could be applied in recon-structing the metabolic network of histidine for biotechnologicalapplications.

5. Conclusion

In this study, we reported the function of an ATP-PRT ina Gram-positive bacterium, C. glutamicum. In vivo genetic studiesdemonstrated that HisGCg is essential for histidine biosynthesis.The recombinant HisGCg, purified by His-tag affinity chromatog-raphy and gel filtration, mainly existed as a hexamer with thesubunit molecular weight of 32191 Da. The enzyme had a higheraffinity for PRPP than ATP with optimal activity at 30 �C and pH

10.0. The inhibitory effect of histidine on HisGCg activity wasdependent on the pH, suggesting that the protonation of thea-amino group of histidine is important for its binding to HisGCg.Based on the homology model and mutational analysis, N215,L231 and T235 in the C-terminal domain of HisGCg were thehistidine binding residues and the mutant N215K/L231F/T235Aresisted to histidine inhibition the most with 37-fold increase in Kivalue. Our results demonstrated that the binding between histi-dine and HisGCg is stabilized by several reciprocal forces and thehydrogen bonds formed with the a-amino group of histidine playa major role. Furthermore, the positive electrostatic potential ofprotein surface responsible for histidine binding enhances theelectrostatic repulsive force to prevent histidine from binding tothe C-terminal domain of HisGCg, resulting in the release of allo-steric inhibition.

Acknowledgments

We thank Dr. Zhenchuan Fan for English revision. We aregrateful to Yong Liang and Qian Liu for critical reading of themanuscript. This work was supported by grants from the Ministryof Science and Technology of China (2008BAI63B01, 2008ZX09401-05 and 2010ZX09401-403) and Beijing Natural Science Foundation(5112023).

Fig. 7. Comparison of the structures and molecular surfaces of wild type HisGCg and the mutant N215K/L231F/T235A. (A) A view of the histidine binding site with residues rep-resented in stick, colored according to atom type. Histidine is shown in ball. Hydrogen bonds formed between histidine and HisGCg are shown as pink dotted lines. (B) molecularsurface of wild type HisGCg and the mutant N215K/L231F/T235A by electrostatic potential. The positive and negative charges calculated using APBS are represented in blue and redwith �5.0 kT contours. Three amino acid residues binding histidine in wild type HisGCg and the mutant N215K/L231F/T235A are labelled. All figures were produced using PyMOL.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)



Appendix. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.biochi.2011.11.015.

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