The Regulatory Mechanism of Mycobacterium tuberculosis

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The Regulatory Mechanism of Mycobacterium tuberculosis Phosphoserine Phosphatase SerB2 Gregory A. Grant Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01082 • Publication Date (Web): 15 Nov 2017 Downloaded from http://pubs.acs.org on November 16, 2017

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The Regulatory Mechanism of Mycobacterium tuberculosis Phosphoserine Phosphatase SerB2 Gregory A. Grant From the Departments of Developmental Biology and Medicine, Washington University School of Medicine, 660 S. Euclid Avenue, Box 8103, St. Louis, Missouri 63110. Running Title: Regulation of M. tuberculosis SerB2 To whom correspondence should be addressed: Prof. Gregory A. Grant, Department of Developmental Biology, Box 8103, Washington University School of Medicine, St. Louis, MO 63110. Phone 314-3623367, FAX 314-362-4698, email [email protected]. Keywords: Allosteric Regulation, Enzyme catalysis, Enzyme kinetics, Phosphoserine phosphatase, serB2, Mycobacterium tuberculosis, ACT domain, L-serine biosynthesis.

ABSTRACT Almost all organisms contain the same biosynthetic pathway for the synthesis of L-serine from the glycolytic intermediate, D-3-phosphoglycerate. However, regulation of this pathway varies from organism to organism. Many organisms control the activity of the first enzyme in the pathway, D-3phosphoglyerate dehydrogenase (PGDH), by feedback inhibition through the interaction of L-serine with the ACT domains within the enzyme. The last enzyme in the pathway, phosphoserine phosphatase (PSP) has also been reported to be inhibited by L-serine. The high degree of sequence homology of M. tuberculosis PSP (mtPSP) with M. avium PSP (maPSP), which has recently been shown to contain ACT domains, suggested that the mtPSP also contained ACT domains. This raised the question of whether the ACT domains in mtPSP played a similar functional role as the ACT domains in PGDH. This investigation reveals that L-serine allosterically inhibits mtPSP by a mechanism of partial competitive inhibition by binding to the ACT domains. Therefore, in mtPSP, L-serine is an allosteric feedback inhibitor that acts by decreasing the affinity of the substrate for the enzyme. mtPGDH is also feedback inhibited by L-serine, but only in the presence of millimolar concentrations of phosphate. Therefore, the inhibition of mtPSP by L-serine

would act as a secondary control point for the regulation of the L-serine biosynthetic pathway

under physiological conditions where the level of phosphate would be below that needed for L-serine feedback inhibition of mtPGDH.

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Biochemistry

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INTRODUCTION L-serine

occupies a pivotal position in metabolism due to its role as a precursor for many essential

metabolites. In addition to incorporation into proteins, L-serine is central to the production of glycine, cysteine, tryptophan, phosphatidyl L-serine, sphingolipids, purines, porphyrins and glyoxylate. As the major precursor to glycine, it also produces the one-carbon unit (C1) that is the donor in methylation reactions mediated by derivatives of tetrahydrofolate and S-adenosyl methionine. Almost all organisms, from bacteria to mammals, contain a pathway for the biosynthesis of L-serine from the glycolytic intermediate, D-3-Phosphoglycerate (PGA) (1-9). NH 3+

O O-

O

O-

NAD+

O

O-

αKG

O

NH 3+

O O

O-

O

O-

O

PGDH

O

O

P O-

O

PHP

O-

O

P

P

PGA

O

O-

O

NH 3+

O-

O-

OO

PO42-

H2O

NADH+ H+

OH

O-

Glu

O-

O

PS

PSAT

H

O

O-

PSP

Ser

Scheme 1. The L-Serine Biosynthetic Pathway. PGA, D-3-phosphoglycerate; PHP, + + NAD+ NADH+HSer, Glu PS phosphohydroxypyruvate; PS, phoshoserine; L-serine; NAD /NADH, nicotinamde adenine dinucleotide; Glu, L -glutamic acid; aKG, alpha-ketoglutarate; PGDH, D-3-phospoglycerate dehydrogenase; PSAT, phosphoserine aminotransferase; PSP, αKG Glu phosphoserine phosphatase. GDH PSAT PHP O

O-

O

NH 3+

O

O-

O

O-

O

O-

O

P

O-

O

O-

O-

O

O

NH4+

The biosynthetic pathway (Scheme 1) consists of three enzymes, D-3-phosphoglycerate PHP

αKG

NAD+

NADH +H+

dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT) and phosphoserine phosphatase (PSP). NH 3+

NH 3+

O-

O

O-

O

O

O-

O

O-

O

P

αKG Glu GDH3, 5-9) by converting PGDHPSinitiates the PSAT entry of PGA into the pathway (2, it to phosphohydroxypyruvate NH4+ O-

O-

O

O

O-

O

(PHP) with the concomitant reduction of NAD+. PHP is then converted to phosphoserine (PS) along with the conversion ofH glutamate (aKG) by PSAT. Lastly, phosphoserine is converted PO4 (Glu) to a-ketoglutarate NH4+ + H2O O 2-

2

NH 3+

O

NH 3+

O-

to L-serine and phosphate in an irreversible reaction by PSP. O

O-

O

O

O-

PS

O

O

P

H

O

O-

PSP

Ser

O-

LSD

Pyr

The regulation of the L-serine biosynthetic pathway differs from organism to organism. For instance, in E. coli the end product of the pathway, L-serine, inhibits the activity of PGDH by binding to ACT domains located in the C-terminal portion of the enzyme. The sensitivity of PGDH to L-serine also varies significantly. For instance, E. coli PGDH has an IC50 for L-serine of approximately 10-20 µM

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while PGDH from Corynebacterium glutamicum has an IC50 of approximately 10 mM (10). In mammals and many other eukaryotes, serine has no effect on the activity of PGDH. Although PGDH from these species often possess ACT domains, they have lost the ability to respond to L-serine due to alterations at the serine binding sites. PGDH from M. tuberculosis displays unusual properties in that it can be inhibited by L-serine at low micromolar levels (IC50 = 35µM), but only in the presence of phosphate ions (11). PSP has also been reported to be inhibited by L-serine, but it is not always clear form the literature if this is due to simple product inhibition at the catalytic site or if there is a specific allosteric “feedback” mechanism. The latter mechanism may be of particular importance in those organisms where PGDH is not feedback regulated. Genes designated serB1 and serB2 are both listed as phosphoserine phosphatases in various databases but serB2 is the only one essential for mycobacterial growth and infectivity in tuberculosis (12, 13). PSP has been crystallized from human (14), M. jannaschii (15, 16), and M. avium (17) and kinetic characteristics reported for the human (18, 19), rat (20), chicken (21), M. tuberculosis (22, 23), H. thermophilis (24), P. gingivalis (25), and P. aeruginosa (26) enzymes. In the few instances where cloning and expression has been attempted for the serB1 gene product, from M. tuberculosis for instance (22), it has proven difficult to express and has not been studied further. Interestingly some, but not all, PSP enzymes contain ACT domains. ACT domains are amino acid binding motifs commonly found in bacterial proteins (27, 28). The prototypic ACT domain was first observed in the structure of E. coli PGDH where it was shown to bind the feedback inhibitor, L-serine, at the distal portions of the interface between two adjacent ACT domains (29). The crystal structure of PSP SerB2 from Mycobacterium avium (maPSP) (17) demonstrated that it was a dimeric enzyme that contains 2 ACT domains per subunit and that the ACT domains of one subunit crossover and interface with the ACT domains of the other subunit (16, Figure 1). However, the structure was that of the apo-form, so Lserine binding to the ACT domains was not directly observed and no kinetic analysis was performed.

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Asp 15

ACT

Asn 123

Gly18

Gly 108

Glu33

ACT

Arg 103

Figure 1. Right: Ribbon diagram of the structure of Mycobacterium avium phosphoserine phosphatase (maPSP, pdb 3p96). The enzyme is a dimer with each subunit consisting of a catalytic domain (dark green and dark blue) and two ACT domains (light green and light blue). The ACT domains, which have a babbab motif, are N-terminal to the catalytic domains and form inter-subunit dimer interfaces very similar to that found in E. coli and M. tuberculosis D-3-phsosphoglycerate dehydrogenases (PGDH). Left: A single ACT domain dimer showing the location of potential L-serine binding residues. The position of two conserved glycine residues is shown in red. PSP from Mycobacterium tuberculosis (mtPSP), which has a high degree of sequence similarity with maPSP, is reported to be very sensitive to L-serine with an IC50 = 0.78 µM, a kcat = 2.4 x104 s-1 and a Km = 136 µM for phosphoserine (22). On the other hand, another study of mtPSP (23) reported a kcat = 0.15 s-1 and a Km = 93 µM. The reason for the large difference in kcat between these two reports is not known. PSP from Porphyromonas gingivalis (pgPSP), which also contains ACT domains, is reported to have a kcat of 25 s-1 and a Km = 2 mM (25). Early reports showed that PSP from chicken and rat liver, which do not contain ACT domains (see Figure 2), were inhibited by L-serine with an IC50 = 0.5mM with the chicken liver enzyme exhibiting apparent uncompetitive inhibition kinetics (20, 21), corresponding to a model where serine does not bind to the free enzyme, but rather to the enzyme-substrate complex. The presence of ACT domains in some phosphoserine phosphatases raises the question of whether or not they modulate the activity of the enzyme. A recent article by Yadav et. al. (22) showed an

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effect on the modulation of activity by L-serine when the ACT domains of mtPSP SerB2 were removed. However, they didn’t measure the serine sensitivity of individual residue mutations and the limited kinetic analysis that was reported did not address the mechanism of this effect but simply attributed it to interaction of L-serine with the ACT domains. The unusual feedback inhibition properties of mtPGDH and the importance of M. tuberculosis as a dangerous pathogen that has developed multi-drug resistance (MDR) and extremely drug resistant (XDR) strains prompted us to better define the enzymes of this pathway, and in particular PSP. Work on the mechanism of PGDH was summarized in a recent review (30), but far less information is available for the other two enzymes in the serine biosynthetic pathway. For this investigation, we have cloned and characterized the gene product of M.tb serB2 (Rv3402c). The results indicate that PSP SerB2 form M. tuberculosis is feedback regulated by an allosteric mechanism of partial competitive inhibition through interaction of L-serine with the enzyme at a site other than the active site. Specific amino acid reside mutations also demonstrate that L-serine inhibits the enzyme through interaction with the ACT domains. We also attempted to clone and express the serB1 (Rv0505c) gene product. However, as reported previously (22), we were not able to express it in stable form and did not study it further.

EXPERIMENTAL PROCEDURES PSP SerB2 from M. tuberculosis (Mtb) was cloned from Mtb DNA (Erdman) by PCR amplification and expressed in Escherichia coli BL21 DE3 cells. The enzyme is expressed with a hexa-histidine tag at the amino terminus by placing it into the BamH1 cloning site of pSV281. The cloning and expression of the enzyme was performed essentially in the same way as that previously reported for D-3-phosphoglycerate dehydrogenase from Mtb (11). Site specific mutagenesis was carried out with a PCR based protocol as previously described (31). The resulting mutated genes were sequenced to verify the presence of the desired mutation and the absence of unwanted mutations. PSP catalyzes the irreversible conversion of phosphoserine to serine and phosphate in the presence of magnesium ion. The reaction was measured in 50 mM MOPS buffer, pH 7.0 and was assayed

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by either following the production of phosphate colorimetrically or the production of L-serine by coupling the reaction to L. pneumophila L-serine dehydratase (lpLSD) (32). L-serine dehydratase was expressed and purified as previously described (32). L-serine dehydratase converts phosphoserine to pyruvate and ammonia and is monitored at 250 nm which detects the appearance of pyruvate (32). The coupled reaction was optimized so that the initial enzyme, PSP, was rate limiting and the rate was determined with the linear portion of the curve where the intermediate, L-serine, reaches a steady state following an initial lag phase. Phosphate ion was determined colorimetrically by modification of the assay of Chen et.al. (33) as reported on-line by Dunham (Dunham.gs.washington.edu/MDphosphateassay.htm). The reaction was allowed to proceed for 30 or 60 seconds which was determined to be a period over which the product formation was linear. Each kinetic analysis was performed in duplicate and representative data are presented for the velocity versus substrate plots. For double reciprocal plots, the replicate mean was plotted with the error bars representing ± SEM. Where error bars are not evident in the plots, they are within the area of the symbol. The double reciprocal plots were fit to a straight line by linear regression analysis. Plots of activity versus phosphoserine concentration showed indications of substrate inhibition so the data was fit to a standard equation for substrate inhibition, v = Vm / (1 + (Km / [S]) + ([S] / Ki))

(Eq. 1).

where v is the velocity at any substrate concentration [S], Vm is the maximum velocity, Km is the Michaelis constant, and Ki is the inhibition constant. The plot of velocity versus substrate at fixed L-serine concentrations were fit to the velocity equation for partial competitive inhibition, [PS]

+

Ks

v= 1+

[PS] Ks

+

[PS] [Ser] aKsKi [Ser] Ki

+

[PS] [Ser] aKsKi

(Eq. 2)

where PS is the substrate, phosphoserine, Ser is the inhibitor, L-serine, and the other parameters are as defined in Scheme 2.

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Biochemistry

The following equation represents the special case where both partial competitive inhibition and pure competitive inhibition are occurring, [PS] Ks

v= 1+

[PS] Ks

+

[Ser] Ki

+

[PS] [Ser]

+

aKsKi

[PS] [Ser] aKsKi

+

[PO4] Ki2

+

[Ser] [PO4] gKi2Ki

(Eq. 3)

In addition to a term for phosphate inhibition, a term for the product of two inhibitors ([Ser][PO4]) is also present. Plots were produced and fit using Kaleidograph from Synergy Software.

RESULTS Structure and Sequence Conservation. The structure of maPSP (Figure 1) was determined by the Seattle Structural Genomics Center for Infectious Disease and deposited in the RCSB Protein Data Bank with the pdb code 3p96. The protein is a dimer with each subunit consisting of a catalytic domain and two ACT domains. The ACT domains are N-terminal to the catalytic domain with the first ACT domain of one subunit forming a dimeric interface with the second ACT domain of the other subunit. An alignment of the MtbPSP SerB2 and MtbPSP SerB1 sequences with the M. avium PSP, shown in Figure 2, demonstrates that MtbPSP SerB2 is most similar to maPSP. Also shown are the ACT domain sequences from E. coli and M. tuberculosis PGDH and, for comparison, the sequence of chicken liver PSP that does not contain ACT domains. The residues critical for serine binding in ecPGDH and mtPGDH are indicated with a plus (+) sign. Critical catalytic residues in the PSP SerB proteins are indicated with an asterisk (*). Conserved areas of sequence are highlighted in yellow. There is very good conservation of sequence at the catalytic residues of MtbPSP SerB2 and MtbPSP SerB1 with M. avium PSP SerB2. The ACT domains, on the other hand, are less well conserved, and MtbPSP SerB1 is missing important segments at the putative serine binding sites that are present in MtbPSP SerB2.

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+ + + + + ecPGDH ACT LMHIHENRPGVLTALNKIFAEQGVNIAAQYLQTSAQMGYVVIDIEADEDVAE mtPGDH ACT LIIHYVDRPGALGKIGTLLGTAGVNIQAAQLSEDAEGPGATILLRLDQDVPD mtserB1 MGLTCWPRTAAGRVHDESRCGLANFDTALGLQINPRQPRAPPRICRIGLITAAASATGmtserB2 MPAKVSVLITVTGMDQPGVTSALFEVLAQHGVELLNVEQVVIRGRLTLGVLVSCPLDVAD maPSP GPGSMNSPPKVSVLITVTGVDQPGVTATLFEVLSRHGVELLNVEQVVIRHRLTLGVLVCCPADVAD ecPGDH ACT mtPGDH ACT mtserB1 mtserB2 maPSP

+ + + KALQAMKAIPGTIRARLLY LMHIHENRPGVLTALNKIFAEQ DVRTAIAAAVDAYKLEVVDLS LIIHYVDRPGALGKIGTLLGTA QAPRLGVMMVSSHLGSPDQ----------------------------------------GTALRDDVAAAIHGVGLDVAIERSDDLPIIRQPSTHTIFVLGRPITAGAFSAVARGVAAL GPALRHDVEAAIRKVGLDVSIERSDDVPIIREPSTHTIFVLGRPITAAAFGAVAREVAAL

ecPGDH ACT mtPGDH ACT mtserB1 mtserB2 maPSP clPSP

+ + GVNIAAQYLQTSAQMGYVVIDIEADEDVAEKALQAMKAIPGTIRARLLY GVNIQAAQLSEDAEGPGATILLRLDQDVPDDVRTAIAAAVDAYKLEVVDLS ---------------AGHVDLASPADPPPPDASASHSPVDMPAPVAAAGSDRQPPIDLTA GVNIDFIRGISDYPVTGLELRVSVPPGCVGPLQIALTKVAAEEHVDVAVEDYGLAWRTKR GVNIDLIRGVSDYPVIGLELRVSVPPGADEALRTALNRVSSEEHVDVAVEDYTLERRAKR MASLLEMKEIFRNAD

mtserB1 mtserB2 maPSP clPSP

* * * AAFFDVDNTLVQGSSAVHFGRGLAARHYFTYRDVLGFLYAQAKFQLLGKENSNDVAAGR LIVFDVDSTLVQGEVIEMLAARAGAQGQVAAITEAAMRGE------------------LIVFDVDSTLVQGEVIEMLAAKAGAEGQVAAITDAAMRGE------------------AVCFDVDSTVIREEGIDELAKFCGVGDAVAEMTRRAMGG-------------------* RKALAFIEGRSVAELVALGEEIYDEIIAD--KIWDGTRELTQMHLDAGQQVWLITATPY LDFAESLQRR-VATLAGLPATVIDDVAEQ-LELMPGARTTIRTLRRLGFRCGVVSGGFR LDFAQSLQQR-VATLAGLPATVIDEVAGQ-LELMPGARTTLRTLRRLGYACGVVSGGFR TVTFKAALTARLGLIRPSYEQVQKLISDNPPQLTPGIRELVNRLHQRGVQVFLVSGGFQ * ELAATIARRLGLTG------ALGESVDGIFTGRLVGEILHGT-GKAHAVRSLAIRETVA RIIEPLARELMLD------FVASEIVDGILTGRVVGPIVDRP-GKAKALRDFASQYNEL RIIEPLAEELMLDYVA---ANELEIVDGTLTGRVVGPIIDRA-GKATALREFA-QRA—SIVEHVALQLNIPTANVFANRLKFYFNGEYAGFDETQPTAESGGKGKVITHLKEQFH-** * GLNLKRCTAYSDSYNDVPMLSLVGTAVAINPDARLRSLARERGWEIRDFRIARKAARIG GVPMEQTVAVGDGANDIDMLGAAGLGIAFNAKPALREVADASLSHPYLDTVLFLLGVTR GVPMAQTVAVGDGANDIDMLAAAGLGIAFNAKPALREVADASLSHPYLDTVLFLLGVTR ---FKKVVMIGDGATDMEACPPADCFIGFGGNVIRKQVKEKAKWYITHFDELLKELEER

mtserB1 mtserB2 maPSP

VPSALALGAAGGALAALASRRQSR GEIEAADAGDCGVRRVEIPAD GEIEAADAIDGEVRRVEIPPE

mtserB1 mtserB2 maPSP clPSP mtserB1 mtserB2 maPSP clPSP mtserB1 mtserB2 maPSP clPSP

60

120

180

240

300

360

420

Figure 2. Sequence alignment of M. avium phosphoserine phosphatase (maPSP), with serB1 and serB2 from M. tuberculosis (mtserB1 and mtserB2). The sequence of the ACT domains from E. coli PGDH (ecPGDH ACT) and M. tuberculosis PGDH (mtPGDH ACT) are also shown for identification of the putative L-serine binding sites (in Italics and underlined). The sequence of chicken liver PSP (clPSP) which does not contain ACT domains is also shown for comparison. Residues critical for in L-serine binding in PGDH are marked with a +. Active site residues in phosphoserine phosphatases are marked with a *. Areas of conserved sequence are highlighted in yellow.

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Biochemistry

Kinetic Analyses of Native Mtb serB2. Plots of velocity versus substrate concentration for native MtbPSP SerB2 are shown in Figure 3 and the kinetic parameters derived from fitting the data to equation 1 are shown in Table 1.

Figure 3. Activity versus substrate (PS, phosphoserine) concentration plots for native mtPSP SerB2. Data from the coupled assay with L-serine dehydratase, which monitors L-serine production, is on the left and data from the colorimetric assay which monitors phosphate production is on the right. The data are fit to equation 1. Table 1 Kinetic Parameters of Native PSP Enzyme (assayb)

Km (mM)

kcat (s-1)

kcat / Km (M-1s-1)

Ki,Pser a (mM)

4.9 x 104

23 ± 5 0.019 ± 0.002

Native (PO4)b

0.38 ± 0.05 18.6 ± 1.1

Native (LSD)b

2.5 ± 0.2

11.8 ± 0.5 0.47 x 104

D15AE33A (PO4)

0.68 ± 0.2

20.5 ± 2.9

3.0 x 104

13 ± 4

D15AE33A (LSD) 0.71 ± 0.12 17.9 ± 1.2

2.5 x 104

86 ± 27

G18AG108A (PO4) 0.15 ± 0.02 15.2 ± 0.4

10 x 104

NPc

Ki,Ser (mM)

IC50,ser (mM) 0.20 ± 0.02

97 ± 24

a

IC50,PO4 (mM)

40 ± 3 6.7

3.3 ± 0.2 83 ± 6

NDd

1.1 ± 0.1

Fit to equation 1.b PO4, phosphate colorimetric assay; LSD, enzyme coupled assay. c NP, substrate inhibition is not present. d ND, not determined.

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The left-hand plot shows the data obtained with the coupled assay using L-serine dehydratase to detect the serine produced while the right-hand plot shows the data from a colorimetric assay that measures the phosphate produced. It is noteworthy that the two assays, both using the same substrate and buffer mix, produce different results. However, this can be explained by considering the nature of the respective assays. First, the Km value for the colorimetric phosphate assay is approximately 7-fold lower and the kcat/Km value is 10-fold higher when compared to the enzyme coupled assay. The major difference between the two assays is that serine reaches a steady state concentration in the coupled assay while its concentration is comparatively very low under the initial velocity conditions of the phosphate assay. This suggests that the presence of serine in the assay is exerting an inhibitory effect. In particular, the kcat/Km value is essentially a second order rate constant for substrate binding, so its decrease when L-serine is present indicates an effect on substrate binding. Second, there is more apparent substrate inhibition with the phosphate color assay than the coupled assay, suggesting that the steady state level of serine in the coupled assay is decreasing the unproductive interaction of the enzyme with phosphoserine. To study this mechanism further, inhibition analysis was performed by determining the activity of the enzyme in the presence of the products, L-serine and phosphate. The phosphate colorimetric assay was used when serine was varied and the dehydratase coupled assay was used when phosphate was varied. Since the reaction catalyzed by PSP is an irreversible Uni-Bi reaction, both products would be expected to act as simple dead-end competitive inhibitors at the active site unless there was a second binding site present. The results are shown as double reciprocal plots in Figures 4. When serine concentration is varied (Figure 4, left), the lines intersect at the 1/v axis. When phosphate concentration is varied (Figure 4, right) the lines intersect near the 1/v axis. A replot of the slopes from the double reciprocal plot versus inhibitor concentration is shown in Figure 5. Neither plot

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Biochemistry

Figure 4. Kinetic analysis of native mtPSP SerB2. Left: Double reciprocal plot of activity measured at various phosphoserine concentrations at varied fixed concentrations of L-serine using the colorimetric assay for phosphate. L-serine concentrations are 0 mM (●), 0.05 mM (■), 0.1 mM (◆), 0.25 mM (▲), and 0.5 mM (▼). Right: Double reciprocal plot of activity measured at various phosphoserine concentrations at varied fixed concentrations of phosphate using the coupled assay for L-serine. Phosphate concentrations are 0 mM (●), 25 mM (■), 50 mM (◆), 100 mM (▲), and 125 mM (▼). Data were fit to the equation for a straight line using linear regression analysis and error bars represent ± SEM. If error bars are not evident, they are within the area of the symbol.

Figure 5. Left: Slope versus L-serine concentration replot of data from the double reciprocal plot. Right: Slope versus phosphate concentration replot of data from the double reciprocal plot. The replots are fit using the smooth curve function of Kaleidograph.

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is linear, with the replot for varied serine being hyperbolic and that for varied phosphate being parabolic. Replots of the velocity as a function of inhibitor concentration at constant substrate concentration are shown in Figure 6.

Figure 6. Left: Plot of activity versus inhibitor (L-serine) concentration at constant substrate (phosphoserine) concentrations. The replots are fit using the smooth curve function of Kaleidograph. Right: Plot of activity versus inhibitor (phosphate) concentration at constant substrate (phosphoserine) concentrations. The replots are fit to a straight line by linear regression analysis.

The replots for varied serine plateau at a finite velocity while those for phosphate are linear and extrapolate to zero. While the double reciprocal plots are consistent with a mechanism of competitive inhibition, the replots indicate a difference between the mode of inhibition of the two inhibitors. The hyperbolic slope replot, and the inability of increasing inhibitor concentration to drive the velocity to zero, is indicative of partial competitive inhibition. The classical model for partial inhibition as shown in Scheme 2 (34).

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Biochemistry

E + PS

Ks

E•PS

+

+

Ser

Ser

Ki Ser•E + PS

a Ks

a Ki

Ser•E•PS

kp

b kp

E + P

Ser•E + P

Scheme 2. General diagram of binding equilibria for partial inhibition. Abbreviations are E, enzyme; PS, phosphoserine; P, product; and Ser, serine. When placed to the right of E, it designates binding at the active site. When placed to the left of E, it designates binding at the allosteric site. The substrate and inhibitor bind to the enzyme at different sites to yield ES, EI, and ESI complexes shown as E·PS, Ser·E, and Ser·E·PS in Scheme 2, respectively. If the substrate binds at the active site of the free enzyme with greater affinity than to the EI complex and the ES and ESI complexes both turn over at the same rate, partial competitive inhibition results. In this case, 1 < a < ∞ and b = 1. Pure competitive inhibition would yield linear replots. On the other hand, a parabolic slope replot, as seen when phosphate is varied, is indicative of a mechanism where an inhibitor or two different inhibitors can bind at two different sites on the enzyme. Since serine, a partial competitive inhibitor, is present as well as phosphate, because of the nature of the coupled assay, both appear to be binding to the enzyme to form an EI2 complex to cause inhibition. In this case, in addition to a parabolic slope replot, the lines in a double reciprocal plot would not be expected to have a common intersection point (34). This is what is seen in Figure 4, right. Furthermore, the conditions present in the coupled assay, that is the presence of two inhibitors, provides a situation that is, in effect, a multiple inhibition analysis. A Dixon plot (Figure 7) where 1/v is plotted as a function of one inhibitor concentration in the presence of the other, provides evidence that the two inhibitor binding sites are not mutually exclusive since the lines are intersecting instead of parallel, the latter being the case for mutually exclusive inhibitor binding (34).

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70

60

50

40

1/v 30

20

10

0 -50

0

50

100

150

[PO ] mM 4

Figure 7. Dixon plot of the data from the coupled assay varying phosphate concentrations at fixed concentrations of substrate in the presence of the other inhibitor. Data for 2, (●); 4 (▲); and 10 (x) mM phosphoserine are shown. The Km values derived graphically from the double reciprocal plots in the presence (coupled assay) and absence (phosphate colorimetric assay) of serine are approximately 3.3 mM and 0.4 mM, respectively. These values agree well with what was determined from the velocity vs substrate concentration plots (Figure 3 and Table 1). The IC50 values at equimolar substrate concentrations for Lserine and phosphate are 0.2 mM and 40 mM, respectively. Furthermore, the value for a, determined graphically is approximately 33. Using this value, a fit of the velocity versus substrate concentration curves determined at fixed levels of serine to the velocity equation for partial competitive inhibition (equation 2) yields a Ki for serine = 19 ± 2 µM (Figure 8). Determined graphically, the Ki for serine is approximately 16 µM.

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Biochemistry

0.5

0.4

0.3

v 0.2

0.1

0 0

0.5

1

1.5

2

2.5

[PS] mM

Figure 8. Plot of velocity versus substrate concentration at constant fixed inhibitor concentrations for the colorimetric assay fit to equation 2. L-serine concentrations are 0.05, (■); 0.1, (◆); 0.25, (▲); and 0.5, (▼) mM. Error bars represent ± SEM. If error bars are not evident, they are within the area of the symbol.

Taken as a whole, the data indicate that L-serine is acting as an allosteric inhibitor while phosphate is acting as a simple dead-end product inhibitor. The situation where both inhibitors are present at the same time (coupled assay) is shown in Scheme 3, where the EI2 complex is represented as Ser·E·PO4. Scheme 3 illustrates that increasing phosphate can drive all of the enzyme to the inactive E·PO4 and Ser·E·PO4complexes consistent with what is observed in Figure 6, right. The presence of these two complexes introduce additional terms into the velocity equation (Equation 3). However, its complexity does not allow it to yield a Ki for phosphate because it contains the unknown dependent variable g and the concentration of serine in the coupled assay is unknown.

15


Biochemistry

E•PO4

Ki2

PO4 + E + PS

+ Ser

gKi Ser•E•PO4

gKi2

PO4

Ks

E•PS

+

+

Ser

Ser

Ki +

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Ser•E + PS

aKs

aKi

Ser•E•PS

kp

bkp

E + P

Ser•E + P

Scheme 3. General diagram of binding equilibria for partial inhibition by one inhibitor and competitive inhibition by a second inhibitor. Abbreviations are E, enzyme; PS, phosphoserine; P, product; Ser, Lserine; and PO4, phosphate. When placed to the right of E, it designates binding at the active site. When placed to the left of E, it designates binding at the allosteric site. Production and Kinetic Analyses of ACT domain mutations. In order to probe the mechanism further, two site-directed mutated enzymes were produced. One, named G18AG108A converts conserved glycine residues at position 18 and 108, required for efficient ligand binding in E. coli PGDH (22, 28), to alanine residues whose side chains should interfere with ligand binding. The other converts Asp15 and Glu33 to alanine residues to produce a mutated enzyme labeled D15AE33A. The location of these residues is shown in Figure 1 and they were chosen on the basis of homology to two of the L-serine binding residues in the ACT domain of E. coli PGDH. As can be seen in Figure 1, the mutation of both Asp15 and Glu33 should interfere with serine binding at both sites in an ACT dimer. The double reciprocal plot of the kinetic analysis of G18AG108A is shown in Figure 9, left. Unlike the native enzyme, the lines intersect to the right of the y-axis and above the x-axis. In the case of partial competitive inhibition defined by equation 2, the intersection points are a function of the values of a and b in scheme 2 (34). Intersection of the lines to the right of the y-axis generally occurs when b is greater than 1. In order to demonstrate this, a simulation of equation 2 with the same parameters as Figure 4, left, except that b is now = 5 rather than 1 is shown in Figure 9, right. This simulation approximates the experimental data very closely and demonstrates that the effect of this mutation is to increase the rate of

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Biochemistry

turnover of the Ser·E·PS complex. In effect, while serine can still bind in some manner, the mutations affect the mode of binding in such a way that it relieves to some degree the inhibition of the rate of turnover of the complex. This observation also supports the partial inhibition mechanism shown in scheme 2.

Figure 9. Kinetic analysis of G18AG108A mtPSP SerB2. Left: Double reciprocal plot of activity measured at various phosphoserine concentrations at varied fixed concentrations of L-serine using the colorimetric assay for phosphate. L-serine concentrations are 0 mM (●), 0.5 mM (■), 1 mM (◆), 1.5 mM (▲), 2.5 mM (▼) and 5 mM (x). Data were fit to the equation for a straight line using linear regression analysis and error bars represent ± SEM. If error bars are not evident, they are within the area of the symbol. Right: Simulation of a double reciprocal plot of activity measured at various phosphoserine concentrations at varied fixed concentrations of phosphate using Equation 2. Parameters are left, a=33, b=1; right, a=33, b=5.

The double reciprocal plot of the kinetic analysis of D15AE33A is shown in Figure 10, left and the slope replot is shown on the right. In this case, the lines intersect at the y-axis but the slope replot is linear, indicating pure competitive inhibition. The Ki for L-serine, determined from the slope replot, is approximately 6.7 mM. This is in contrast to a Ki of 0.019 mM for the native enzyme (see Table). In addition, the IC50 for L-serine increases approximately 17 fold, from 0.2 mM to 3.3 mM for the mutation

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of these ACT domain residues. It is also noteworthy that the Km values of the enzyme with both the phosphate colorimetric assay and the enzyme coupled assay are now very similar and comparable to that for the native enzyme in the case of the phosphate color assay which is absent of serine influence. This is additional evidence that the effect of L-serine inhibition has been eliminated by this mutation. Thus, mutation of two of the residues proposed to be involved in L-serine binding to the ACT domain by homology to the PGDH ACT domain produces the anticipated effect of greatly decreasing the sensitivity of the enzyme to inhibition by L-serine.

Figure 10. Kinetic analysis of D15AE33A mtPSP SerB2. Left: Double reciprocal plot of activity measured at various phosphoserine concentrations at varied fixed concentrations of L-serine using the colorimetric assay for phosphate. L-serine concentrations are 0 mM (●), 1.25 mM (■), 2.5 mM (◆), 5 mM (▲), 7.5 mM (▼) and 10 mM (x). Data were fit to the equation for a straight line using linear regression analysis and error bars represent ± SEM. If error bars are not evident, they are within the area of the symbol. Right: Slope replot of the data on the left.

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Biochemistry

DISCUSSION L-serine,

the product of phosphoserine phosphatase, has been reported to be an inhibitor of the

enzyme (20-22) in some species. Whether L-serine functions simply as a product inhibitor by binding competitively to the active site or as an allosteric effector by binding to another site on the enzyme is a question that has not been adequately addressed. Many phosphoserine phosphatases, particularly from mammalian sources, appear to be made up of only a catalytic domain (see Figure 2). However, the crystal structure of M. avium PSP SerB2 revealed that it possesses well conserved ACT domain motifs and the conservation of sequence in M. tuberculosis PSP SerB2 suggests that it does also. Therefore, the presence of ACT domains in mtPSP SerB2 raises the question of whether or not they are functional in modulating the activity of the enzyme. This investigation shows that there is an allosteric L-serine binding site in mtPSP SerB2 in the ACT domains as suggested by Yadev (22). The data indicate that serine acts as a partial competitive inhibitor. The parabolic nature of the coupled enzyme assay slope replot, where both L-serine and phosphate are present, indicates that there is a second inhibitor binding site. The intersecting nature of the Dixon plot shows that the two inhibitors can bind simultaneously. The high IC50 for phosphate relative to that for L-serine and the ability of phosphate to drive the velocity to zero indicates that it is acting as a classical dead-end competitive inhibitor, consistent with an irreversible enzyme reaction where product cannot be converted back to substrate. Theoretically, L-serine could also act as a dead-end competitive inhibitor. However, this does not appear to be manifest at the low level of serine present under the conditions used for the reactions when phosphate production is detected until the mutations at Asp15 and Glu33 are made. Control of L-serine biosynthesis in bacteria is critical for their survival. High levels of L-serine are toxic to most bacteria because L-serine at high levels can interfere with basic cellular processes and inhibit certain other critical enzymes (35). It has been reported that in E. coli, as much as 15% of glucose carbon goes through L-serine on its way to other metabolites (36). Therefore, control of the pathway is critical to conserve the glycolytic intermediate, D-3-phosphoglyerate, that is the precursor to L-serine as

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well as pyruvate that enters the TCA cycle. E. coli PGDH is very sensitive to L-serine as a feedback inhibitor and this probably provides sufficient control of the pathway in this species. However, in those organisms where this control point is missing or its impact is lessened, control at other enzymes along the pathway can play an important role. This is likely the case for M. tuberculosis because the sensitivity of its PGDH to L-serine feedback control is dependent on cellular conditions. In vitro, mtPGDH is inhibited at low micromolar levels of L-serine only in the presence of relatively high concentrations (30-50 mM) of phosphate (11). If these characteristics translate to the in vivo situation and phosphate is lacking intracellularly during certain stages of the M. tuberculosis life cycle, control of the pathway at some other point will be necessary. This study, regarding the mechanism of L-serine interaction with PSP, provides evidence for better understanding how PSP can act as a secondary control point in M. tuberculosis and perhaps as a primary control point in other organisms.

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Biochemistry

ACKNOWLEDGMENT The author thanks Xiao Lan Xu for excellent technical assistance. Funding was provided by the Department of Developmental Biology, Washington University School of Medicine. CONFLICT OF INTEREST

The author declares that he has no conflicts of interest with the contents of this article. AUTHOR CONTRIBUTIONS GAG conceived and directed the study and wrote the paper

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REFERENCES 1. Sallach, H. J. (1956) Formation of serine hydroxypyruvate and L-alanine. J. Biol. Chem. 223, 11011108. 2. Greenberg, D. M., and Ichihara, A. (1957) Further studies on the pathway of serine formation from carbohydrate. J. Biol. Chem. 224, 331- 340. 3. Hanford, J. and Davies, D. D. (1958) Formation of phosphoserine from 3-phosphoglycerate in higher plants. Nature, 182, 532- 533. 4. Willis, J. E. and Sallach, H. J. (1962) Evidence for a mammalian D-glyceric dehydrogenase. J. Biol. Chem. 237, 910- 915. 5. Willis, J. E. and Sallach, H. J. (1964) The occurrence of D-3-phosphoglycerate in animal tissue. Biochim. et Biophys. Acta 81, 39-54. 6. Walsh, D. A., and Sallach, H. J. (1966) Comparitive studies on the pathways for serine biosynthesis in animal tissues. J. Biol. Chem. 241, 4068-4076. 7. Cheung, G. P., Rosenblum, I. Y., and Sallach, H. J. (1968) Comparitive studies of enzymes related to serine metabolism in higher plants. Plant Physiol. 43, 1813-1820. 8. Nelson, D. L., Cox, M. M., and Lehninger, A. (2009) Lehninger: Principles of Biochemistry, 5th Edition, W. H. Freeman, New York. 9. Voet, D. and Voet, J. G. (2011) Biochemistry, 4th Edition, John Wiley & Sons, Hoboken, New Jersey. 10. Peters-Wendisch, P., Netzer, R., Eggeling, L., and Sahm, H. (2002) 3-Phosphoglycerate dehydrogenase from Corynebacterium glutamicum: the C-terminal domain is not essential for activity but is required for inhibition by L-serine. Appl. Microbiol. Biotechnol. 60, 437-441. 11. Xu, X. L., and Grant, G. A. (2014) Regulation of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase by phosphate-modulated quaternary structure dynamics and a potential role for polyphosphate in enzyme regulation. Biochemistry 53, 4239-4249. 12. Sassetti, C. M., Boyd, D. H., and Rubin, E. J. (2003) Genes required for mycobacterial growth defined by high-density mutagenesis. Mol. Microbiol. 48, 77-84. 13. Sassetti, C. M., and Rubin, E. J. (2003) Genetic requirements for mycobacterial survival during infection. Proc. Natl. Acad. Sci. U.S.A. 100, 12989-12994. 14. Kim, H-Y., Yong-Seok Heo, Y-S., Kim, J. H., Park, M. H, Moon, J., Kim, E., Kwon, D., Yoon, J., Shin, D., Jeong, E-j., Park, S. Y., Lee, T. G., Jeon, Y. H., Ro, S., Cho, J. M. and Hwang, K. Y. (2002) Molecular basis for the local conformational rearrangement of human phosphoserine phosphatase. J. Biol. Chem. 277, 46651-46658. 15. Wang, W., Kim, R., Jancarik, J., Yokota, H. and Kim, S-H. (2001) Crystal structure of phosphoserine phosphatase from Mathanococcus jannaschii, a hyperthermophile, at 1.8 Å resolution. Structure

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9, 65-71. 16. Wang, W., ho, S. H., Kim, R., Jancarik, J., Yokota, H. Nguyen, H. H., Grigoriev, I. V., Wemmer, D. E. and Kim, S-H. (2002) Structural Characterization of the Reaction Pathway in Phosphoserine Phosphatase: Crystallographic “snapshots” of Intermediate States. J. Mol. Biol. 319, 421-431. 17. Abendroth, J., Gardberg, A.S., Robinson, J. I., Chritensen, J. S., Staker, B. L. (2011) SAD phasing using iodide ions in a high throughput structural genomics environment. J. Struct. Funct. Genomics, 12, 83-95. 18. Collet, J-F., Gerin, I., Rider, M. H., Viega-da-Cunha, M. and Van Schaftingen, E. (1997) Human l-3phosphoserine phosphatase: sequence, expression, and evidence for a phosphoenzyme intermediate. FEBS Lett. 408, 281-284. 19. Collet, J-F., Stroobant, V. and Van Schaftingen, E. (1999) Mechanistic studies of phosphoserine phosphatase, an enzyme related to P-type ATPases. J. Biol. Chem. 274, 33985-33990. 20. Borkenhagen, L. F., and Kennedy, E. P. (1959) The enzymatic exchange of l-serine with O-phosphol-serine catalyzed by a specific phosphatase. J. Biol. Chem. 234, 849-853. 21. Neuhaus, F. C., abd Byrne, W. L. (1960) Metabolism of Phosphoserine. III. Mechanism of OPhosphoserine Phosphatase. J. Biol. Chem. 235, 2019-2024. 22. Yadav, G. P., Shree, S., Maurya, R., Rai, N., Singh, D. K., Srivastava, K. K., and Ramachandran, R. (2014) Characterization of M. tuberculosis ser B2, an essential HAD-family phosphatase, reveals novel properties. PLoS ONE 9(12): e115409. doi:10.1371/journal.pone. 0115409. 23. Arora, G., Tiwari, P., Mandal, R. S., Gupta, A., Sharma, D., Saha, S., and Singh, R. (2014) High throughput screen identifies small molecule inhibitors for Mycobacterium tuberculosis Phosphoserine phosphatase. J. Biol. Chem. 289, 25149-25165. 24. Chiba, Y., Oshima, K., Arai, H., Ishii, M., and Igarashi, Y. ( 2012) Discovery and analysis of cofactor-dependent phosphoglycerate mutase homologs as novel phosphoserine phosphatases in Hydrogenobacter thermophilis. J. Biol. Chem. 287, 11934-11941. 25. Tribble, G. D., Mao, S., James, C. E., and Lamont, R. J. (2006) A Porphyromonas gingivalis haloacid dehalogenase family phosphatase interacts with human phosphoproteins and is important for invasion. Proc. Natl. Acad. Sci. USA 103, 11027-11032. 26. Singh, S. K., Yang, K., Karthikeyan, S., Huynh, T., Zhang, X., Phillips, M. A., and Zhang, H. (2004) The thrH gene product of Pseudomonas aeruginosa is a dual activity enzyme with a novel phosphoserine:homoserine phosphotransferase activity. J. Biol. Chem. 279, 13166-13173. 27. Chipman, D. M., and Shaanan, B. (2001) The ACT Domain Family. Curr. Opin. Struct. Biol. 11, 694700. 28. Grant, G. A. (2006) The ACT Domain: A Small Molecule Binding Domain and its Role as a Common Regulatory Element. J. Biol. Chem. 281, 33825-33829. 29. Schuller, D., Grant, G. A., and Banaszak, L. (1995) Crystal Structure Reveals the Allosteric Ligand Site in the Vmax -type Cooperative Enzyme: D-3-Phosphoglycerate Dehydrogenase. Nature

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Structural Biology 2, 69-76. 30. Grant, G. A. (2012) Contrasting catalytic and allosteric mechanisms for phosphoglycerate dehydrogenases. Arch. Biochem. Biophys. 519, 175-185. 31. Dey, S., Burton, R. L., Grant, G. A., and Sacchettini, J. C. (2008) Structural analysis of substrate and effector binding in Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogense. Biochemistry 47, 8271-8282. 32. Xu, X. L., Chen, S., and Grant, G. A. (2011) Kinetic, mutagenic, and structural homology analysis of L-serine dehydratase from Legionella pneumophila. Arch. Biochem. Biophys. 515, 28-36. 33. Chen, P. S. Jr., Toribara, T. Y., and Warner, H. (1956) Microdetermination of phosphorous. Anal. Chem. 28, 1756-1758. 34. 34. Segel, I. H. (1975) Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady State Enzyme Systems. John Wiley and Sons, New York. 35. Mundhada, H., Seoane, J. M., Schneider, K., Koza, A., Christensen, H. B., Klein, T., Phaneuf, P. V., Herrgard, M., Feist, A. M., and Nielsen, A. T. (2017) Increased production of L-serine in Escherichia coli through adaptive laboratory evolution. Metabolic Engineering 39, 141-150. 36. Pizer, L. I., and Potochny, M. L. (1964) Nutritional and regulatory aspects of serine metabolism in Escherichia coli. J. Bacteriol. 88, 611-619.

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Biochemistry

Asp 15

ACT

Asn 123

Gly18

Gly 108

Glu33

ACT

Arg 103


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

+ + + + + ecPGDH ACT LMHIHENRPGVLTALNKIFAEQGVNIAAQYLQTSAQMGYVVIDIEADEDVAE mtPGDH ACT LIIHYVDRPGALGKIGTLLGTAGVNIQAAQLSEDAEGPGATILLRLDQDVPD mtserB1 MGLTCWPRTAAGRVHDESRCGLANFDTALGLQINPRQPRAPPRICRIGLITAAASATGmtserB2 MPAKVSVLITVTGMDQPGVTSALFEVLAQHGVELLNVEQVVIRGRLTLGVLVSCPLDVAD maPSP GPGSMNSPPKVSVLITVTGVDQPGVTATLFEVLSRHGVELLNVEQVVIRHRLTLGVLVCCPADVAD ecPGDH ACT mtPGDH ACT mtserB1 mtserB2 maPSP

+ + + KALQAMKAIPGTIRARLLY LMHIHENRPGVLTALNKIFAEQ DVRTAIAAAVDAYKLEVVDLS LIIHYVDRPGALGKIGTLLGTA QAPRLGVMMVSSHLGSPDQ----------------------------------------GTALRDDVAAAIHGVGLDVAIERSDDLPIIRQPSTHTIFVLGRPITAGAFSAVARGVAAL GPALRHDVEAAIRKVGLDVSIERSDDVPIIREPSTHTIFVLGRPITAAAFGAVAREVAAL

ecPGDH ACT mtPGDH ACT mtserB1 mtserB2 maPSP clPSP

+ + GVNIAAQYLQTSAQMGYVVIDIEADEDVAEKALQAMKAIPGTIRARLLY GVNIQAAQLSEDAEGPGATILLRLDQDVPDDVRTAIAAAVDAYKLEVVDLS ---------------AGHVDLASPADPPPPDASASHSPVDMPAPVAAAGSDRQPPIDLTA GVNIDFIRGISDYPVTGLELRVSVPPGCVGPLQIALTKVAAEEHVDVAVEDYGLAWRTKR GVNIDLIRGVSDYPVIGLELRVSVPPGADEALRTALNRVSSEEHVDVAVEDYTLERRAKR MASLLEMKEIFRNAD

mtserB1 mtserB2 maPSP clPSP

* * * AAFFDVDNTLVQGSSAVHFGRGLAARHYFTYRDVLGFLYAQAKFQLLGKENSNDVAAGR LIVFDVDSTLVQGEVIEMLAARAGAQGQVAAITEAAMRGE------------------LIVFDVDSTLVQGEVIEMLAAKAGAEGQVAAITDAAMRGE------------------AVCFDVDSTVIREEGIDELAKFCGVGDAVAEMTRRAMGG-------------------* RKALAFIEGRSVAELVALGEEIYDEIIAD--KIWDGTRELTQMHLDAGQQVWLITATPY LDFAESLQRR-VATLAGLPATVIDDVAEQ-LELMPGARTTIRTLRRLGFRCGVVSGGFR LDFAQSLQQR-VATLAGLPATVIDEVAGQ-LELMPGARTTLRTLRRLGYACGVVSGGFR TVTFKAALTARLGLIRPSYEQVQKLISDNPPQLTPGIRELVNRLHQRGVQVFLVSGGFQ * ELAATIARRLGLTG------ALGESVDGIFTGRLVGEILHGT-GKAHAVRSLAIRETVA RIIEPLARELMLD------FVASEIVDGILTGRVVGPIVDRP-GKAKALRDFASQYNEL RIIEPLAEELMLDYVA---ANELEIVDGTLTGRVVGPIIDRA-GKATALREFA-QRA—SIVEHVALQLNIPTANVFANRLKFYFNGEYAGFDETQPTAESGGKGKVITHLKEQFH-** * GLNLKRCTAYSDSYNDVPMLSLVGTAVAINPDARLRSLARERGWEIRDFRIARKAARIG GVPMEQTVAVGDGANDIDMLGAAGLGIAFNAKPALREVADASLSHPYLDTVLFLLGVTR GVPMAQTVAVGDGANDIDMLAAAGLGIAFNAKPALREVADASLSHPYLDTVLFLLGVTR ---FKKVVMIGDGATDMEACPPADCFIGFGGNVIRKQVKEKAKWYITHFDELLKELEER

mtserB1 mtserB2 maPSP

VPSALALGAAGGALAALASRRQSR GEIEAADAGDCGVRRVEIPAD GEIEAADAIDGEVRRVEIPPE

mtserB1 mtserB2 maPSP clPSP mtserB1 mtserB2 maPSP clPSP mtserB1 mtserB2 maPSP clPSP


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800

600

uM/min

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400

200

0 -5

0

5

10

15

[PS] mM


20

25

30

Biochemistry

80

70

60

50

uM/min

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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/min)

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1/v ( 1/ A

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30

20

10

0 -4

-3

-2

-1

0

1

1/ [PS] mM


2

3

4

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120

250

/min)

100

1/v (1/A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0 -0.4

-0.2

0

0.2

0.4

0.6

1/ [PS] mM


0.8

1

1.2

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14

12

10

slope

8

6

4

2

0 -0.2

0

0.2

0.4

0.6

[Serine] mM


0.8

1

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20

10 -20

0

20

40

60

80

[PO ] mM 4


100

120

140

Page 33 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

0.5

0.4

0.3

v 0.2

0.1

0 -0.1

0

0.1

0.2

0.3

[serine] mM


0.4

0.5

0.6

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 45

0.14

0.12

0.1

0.08

v 0.06

0.04

0.02

0 0

50

100

150

[PO ] mM 4


200

250

Page 35 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

70

60

50

40

1/v 30

20

10

0 -50

0

50

[PO ] mM 4


100

150

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 45

0.5

0.4

0.3

v 0.2

0.1

0 0

0.5

1

1.5

[PS] mM


2

2.5

Page 37 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

12

10

8

1/v

6

4

2

0 -1

-0.5

0

0.5

1/ [PS]


1

1.5

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 45

12

10

8

1/v

6

4

2

0 -1

-0.5

0

0.5

1/[PS]


1

1.5

Page 39 of 45

7

820

/min)

6

1/v (1/A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

5

4

3

2

1

0 -0.5

0

1/ [PS] mM


0.5

1

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 45

7

6

5

4

slope 3

2

1

0 -5

0

5

[Serine] mM


10

15

Page 41 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

NH 3+

O O-

O

O-

NAD+

O

O-

αKG

O

NH 3+

O O

O-

O

O-

O

PGDH

Glu

O O

NADH+H+

NAD+

O-

PSP

O-

O

O-

O

P O-

O

PHP

PSAT

αKG

O-

PHP

αKG

Glu

O

NADH +H+ O

O-

O

O-

O

NAD+

NH 3+

OO

GDH

O

O-

NH4+

NH 3+

O-

O

P O-

PSAT

O

PS

O-

O-

PS

αKG

NH4+

NH4+ + H2O O

O O

O

P O-

GDH

O-

NH 3+

OO

O

PO42-

H2O NH 3+ O

Glu

H

O

O-

PSP

Ser

O H

O

NH 3+

O O-

O

PS

PSAT

PS

O-

O

P O-

O

PHP

O-

O

P

P

PGA

O

O-

O

NH 3+

O-

O-

OO

PO42-

H2O

NADH+ H+

OH

O-

Glu

O-

O

O-

LSD

Pyr


O

Ser

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

E + PS

Ks

E•PS

+

+

Ser

Ser

Ki Ser•E + PS

aKs

aKi

Ser•E•PS

Page 42 of 45

kp

bkp


E + P

Ser•E + P

Page 43 of 45

E•PO4

Ki2

PO4 + E + PS

+ Ser

gKi Ser•E•PO4

gKi2

PO4

Ks

E•PS

+

+

Ser

Ser

Ki +

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Biochemistry

Ser•E + PS

aKs

aKi

Ser•E•PS


kp

bkp

E + P

Ser•E + P

Biochemistry

0.5 50 0.4

820

/min)

40

1/v ( 1/ A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 44 of 45

0.3 30

v 0.2

20

0.1

10

0 -4

-3

-2

-1

0

1

2

3

4

0 -0.1

1/ [PS] mM

0

0.1

0.2

0.3

[serine] mM


0.4

0.5

0.6

PageBiochemistry 45 of 45

1 2 3 CS4Paragon Plus Environme 5 6

The Regulatory Mechanism of Mycobacterium tuberculosis

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