Some of Its Properties J. Agríe. Food Chem. 34, 995-998
445
446
Argoudelis CJ. (1986) Preparation of Crystalline Pyridoxine 5 -Phosphate and
Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita
447
M, Wanner BL, Mori H. (2006) Construction of Escherichia coli K-12 in-frame,
448
single-gene knockout mutants: the Keio collection. Mol Syst Biol. 2, 2006.0008.
449
Belitsky BR. (2004) Bacillus subtilis GabR, a protein with DNA-binding and
450
aminotransferase domains, is a PLP-dependent transcriptional regulator. J Mol
451
Biol. 340(4):655-64.
452
Listeria monocytogenes. Mol Microbiol. 92(5):1113-28.
453
454
Belitsky BR (2014) Role of PdxR in the activation of vitamin B6 biosynthesis in
Bilski P, Li MY, Ehrenshaft M, Daub ME, Chignell CF. (2000) Vitamin B6
455
(pyridoxine) and its derivatives are efficient singlet oxygen quenchers and
456
potential fungal antioxidants. Photochem Photobiol. 71(2):129-34.
457
pyridoxal phosphate. Biochem. J. 61, 315-323.
458
459
Blakley, R. L. (1969) The biochemistry of folic acid and related pteridines. Front.
Biol. 13, 189-218.
460
461
Blakley, R. L. (1955) The interconversion of serine and glycine: participation of
Burns KE, Xiang Y, Kinsland CL, McLafferty FW, Begley TP. (2005)
462
Reconstitution and biochemical characterization of a new pyridoxal-5'-phosphate
463
biosynthetic pathway. J Am Chem Soc. 127(11):3682-3.
464
Cherepanov PP, Wackernagel W. (1995) Gene disruption in Escherichia coli: TcR
465
and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-
466
resistance determinant. Gene 158, 9-14.
467
Côté JP, French S, Gehrke SS, MacNair CR, Mangat CS, Bharat A, Brown ED
468
(2016) The genome-wide interaction network of nutrient stress genes in
469
Escherichia coli. Mbio 7:e01714–01716.
19
470
Darin N, Reid E, Prunetti L, Samuelsson L, Husain RA, Wilson, M, El Yacoubi
471
B, Footitt E, Chong WK, Wilson LC, Prunty H, Pope S, Heales S, Lascelles K,
472
Champion M, Wassmer E, Veggiotti P, de Crécy-Lagard V, Mills PB, Clayton PT
473
(2016) Mutations in PROSC disrupt cellular pyridoxal phosphate homeostasis and
474
cause vitamin-B6-dependent epilepsy. Am J Hum Genet 99:1325–1337.
475
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes
476
in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–
477
6645.
478
De Felice, M., M. Levinthal, M. Iaccarino, and J. Guardiola. 1979. Growth
479
inhibition as a consequence of antagonism between related amino acids: effect of
480
valine in Escherichia coli K-12. Microbiol. Rev. 4342-58.
481
Eisenstein E. (1991) Cloning, expression, purification, and characterization of
482
biosynthetic threonine deaminase from Escherichia coli. J Biol Chem. 266:5801-
483
7.
484
and evolutionary considerations. Annu Rev Biochem. 73, 383-415.
485
486
Eliot AC, Kirsch JF. (2004) Pyridoxal phosphate enzymes: mechanistic, structural,
Eswaramoorthy S, Gerchman S, Graziano V, Kycia H, Studier FW, Swaminathan
487
S (2003) Structure of a yeast hypothetical protein selected by a structural
488
genomics approach. Acta Crystallogr D Biol Crystallogr 59:127–135.
489
Farrant RD, Walker V, Mills GA, Mellor JM, Langley GJ. (2001) Pyridoxal
490
phosphate de-activation by pyrroline-5-carboxylic acid. Increased risk of vitamin
491
B6 deficiency and seizures in hyperpro-linemia type II. J Biol Chem. 276:15107-
492
15116
493
Fitzpatrick TB, Amrhein N, Kappes B, Macheroux P, Tews I, Raschle T. (2007)
494
Two independent routes of de novo vitamin B6 biosynthesis: not that different
495
after all. Biochem J. 407, 1-13.
496
Glazyrina J, Materne EM, Dreher T, Storm D, Junne S, Adams T, Greller G,
20
497
Neubauer P. (2010) High cell density cultivation and recombinant protein
498
production with Escherichia coli in a rocking-motion-type bioreactor. Microb Cell
499
Fact. 9:42.
500
Ito T, Uozumi N, Nakamura T, Takayama S, Matsuda N, Aiba H, Hemmi H,
501
Yoshimura T (2009) The implication of YggT of Escherichia coli in osmotic
502
regulation. Biosci Biotechnol Biochem 73, 2698–2704.
503
Ito T, Iimori J, Takayama S, Moriyama A, Yamauchi A, Hemmi H, Yoshimura T
504
(2013) Conserved pyridoxal protein that regulates Ile and Val metabolism. J
505
Bacteriol 195:5439–5449.
506
Ito T, Yamauchi A, Hemmi H, Yoshimura T (2016) Ophthalmic acid accumulation
507
in an Escherichia coli mutant lacking the conserved pyridoxal 5’-phosphate-
508
binding protein YggS. J Biosci Bioeng 122:689–693.
509
Ito T, Tokoro M, Hori R, Hemmi H, and Yoshimura T (2018) Production of
510
Ophthalmic Acid Using Engineered Escherichia coli. Appl Environ Microbiol 84.
511
Pii: e02806-17.
512
Ito T, Yamamoto K, Hori R, Yamauchi A, Downs DM, Hemmi H, Yoshimura T.
513
(2019) Conserved Pyridoxal 5'-Phosphate Binding Protein YggS Impacts Amino
514
Acid Metabolism through Pyridoxine 5'-Phosphate in Escherichia coli. Appl
515
Environ Microbiol. pii: AEM.00430-19.
516
Johnstone DL, Al-Shekaili HH, Tarailo-Graovac M, Wolf NI, Ivy AS, Demarest
517
S, Roussel Y, Ciapaite J, van Roermund CWT, Kernohan KD, Kosuta C, Ban K,
518
Ito Y, McBride S, Al-Thihli K, Abdelrahim RA, Koul R, Al Futaisi A, Haaxma
519
CA, Olson H, Sigurdardottir LY, Arnold GL, Gerkes EH, Boon M, Heiner-
520
Fokkema MR, Noble S, Bosma M, Jans J, Koolen DA, Kamsteeg EJ, Drögemöller
521
B, Ross CJ, Majewski J, Cho MT, Begtrup A, Wasserman WW, Bui T, Brimble E,
522
Violante S, Houten SM, Wevers RA, van Faassen M, Kema IP, Lepage N;
523
Care4Rare Canada Consortium, Lines MA, Dyment DA, Wanders RJA,
21
524
Verhoeven-Duif N, Ekker M, Boycott KM, Friedman JM, Pena IA, van Karnebeek
525
CDM. (2019) PLPHP deficiency: clinical, genetic, biochemical, and mechanistic
526
insights. Brain. 142, 542-559.
527
Kikuchi G, Motokawa Y, Yoshida T, Hiraga K. (2008) Glycine cleavage system:
528
reaction mechanism, physiological significance, and hyperglycinemia. Proc Jpn
529
Acad Ser B Phys Biol Sci. 84, 246-63.
530
Kitagawa M, Ara T, Arifuzzaman M, Ioka-Nakamichi T, Inamoto E, Toyonaga H,
531
Mori H. (2005) Complete set of ORF clones of Escherichia coli ASKA library (a
532
complete set of E. coli K-12 ORF archive): unique resources for biological
533
research. DNA Res. 12, 291-9
534
Labella JI, Cantos R, Espinosa J, Forcada-Nadal A, Rubio V, Contreras A. (2017)
535
PipY, a Member of the Conserved COG0325 Family of PLP-Binding Proteins,
536
Expands the Cyanobacterial Nitrogen Regulatory Network. Front Microbiol. 8,
537
1244.
538
Laber B, Maurer W, Scharf S, Stepusin K, Schmidt FS. (1999) Vitamin B6
539
biosynthesis: formation of pyridoxine 5'-phosphate from 4-(phosphohydroxy)-L-
540
threonine and 1-deoxy-D-xylulose-5-phosphate by PdxA and PdxJ protein. FEBS
541
Lett. 449(1):45-8.
542
Lambrecht G, Braun K, Damer M, Ganso M, Hildebrandt C, Ullmann H, Kassack
543
MU, Nickel P. (2002) Structure-activity relationships of suramin and pyridoxal-
544
5'-phosphate derivatives as P2 receptor antagonists. Curr Pharm Des. 8, 2371-99.
545
Lawther RP, Calhoun DH, Adams CW, Hauser CA, Gray J, Hatfield GW. (1981)
546
Molecular basis of valine resistance in Escherichia coli K-12. Proc Natl Acad Sci
547
U S A. 78, 922-5.
548
Leavitt RI, Umbarger HE. (1962) Isoleucine and valine metabolism in
549
Escherichia coli. XI. Valine inhibition of the growth of Escherichia coli strain K-
550
12. J Bacteriol. 83, 624-30.
22
551
utilization in Escherichia coli. J Bacteriol. 118, 905-10.
552
553
Meedel TH, Pizer LI. (1974) Regulation of one-carbon biosynthesis and
Mills PB, Surtees RA, Champion MP, Beesley CE, Dalton N, Scambler PJ, Heales
554
SJ, Briddon A, Scheimberg I, Hoffmann GF, Zschocke J, Clayton PT. (2005)
555
Neonatal epileptic encephalopathy caused by mutations in the PNPO gene
556
encoding pyridox(am)ine 5'-phosphate oxidase. Hum Mol Genet. 14(8):1077-86.
557
with pyridoxine-dependent seizures. Nat Med. 12(3):307-309.
558
559
Mills PB, Struys E, Jakobs C, et al. (2006) Mutations in antiquitin inindividuals
Nagarajan, Lakshmanan, and Reginald K. Storms. (1997) Molecular
560
characterization of GCV3, the Saccharomyces cerevisiae gene coding for the
561
glycine cleavage system hydrogen carrier protein. Journal of Biological
562
Chemistry 272, 4444-4450.
563
Narisawa S., Wennberg C., Millan J.L. (2001) Abnormal vitamin B6 metabolism
564
in alkaline phosphatase knock-out mice causes multiple abnormalities, but not the
565
impaired bone mineralization. J Pathol. 193:125–133.
566
Nichols RJ, Sen S, Choo YJ, Beltrao P, Zietek M, Chaba R, Lee S, Kazmierczak
567
KM, Lee KJ, Wong A, Shales M, Lovett S, Winkler ME, Krogan NJ, Typas A,
568
Gross CA (2011) Phenotypic landscape of a bacterial cell. Cell 144:143–156.
569
dependent enzymes. EMBO Rep. 4, 850-4
570
571
Plamann MD, Rapp WD, Stauffer GV. (1983) Escherichia coli K12 mutants
defective in the glycine cleavage enzyme system. Mol Gen Genet. 192:15-20.
572
573
Percudani R, Peracchi A. (2003) A genomic overview of pyridoxal-phosphate-
Plecko B, Zweier M, Begemann A, Mathis D, Schmitt B, Striano P, Baethmann
574
M, Vari MS, Beccaria F, Zara F, Crowther LM, Joset P, Sticht H, Papuc SM, Rauch
575
A. (2017) Confirmation of mutations in PROSC as a novel cause of vitamin B 6 -
576
dependent epilepsy. J Med Genet. 2017 54, 809-814.
577
Powers SG, Snell EE. (1976) Ketopantoate hydroxymethyltransferase. II.
23
Physical, catalytic, and regulatory properties. J Biol Chem. 251, 3786-93.
578
579
Prunetti L, El Yacoubi B, Schiavon CR, Kirkpatrick E, Huang L, Bailly M, El
580
Badawi-Sidhu M, Harrison K, Gregory JF, Fiehn O, Hanson AD, de Crécy-Lagard
581
V (2016) Evidence that COG0325 proteins are involved in PLP homeostasis.
582
Microbiology 162:694–706.
583
Raschle T, Amrhein N, Fitzpatrick TB. (2005) On the two components of
584
pyridoxal 5'-phosphate synthase from Bacillus subtilis. J Biol Chem.
585
280(37):32291-300.
586
mechanism, structure and regulation. Biochim Biophys Acta. 1814, 1597-608.
587
588
Schirch L, Gross T. (1968) Serine transhydroxymethylase. Identification as the
threonine and allothreonine aldolases. J Biol Chem. 1968 243, 5651-5.
589
590
di Salvo ML, Contestabile R, Safo MK. (2011) Vitamin B6 salvage enzymes:
Schirch
V,
Hopkins
S,
Villar
E,
Angelaccio
S.
(1985)
Serine
591
hydroxymethyltransferase from Escherichia coli: purification and properties. J
592
Bacteriol. 163, 1-7.
593
Shiraku H, Nakashima M, Takeshita S, Khoo CS, Haniffa M, Ch'ng GS, Takada
594
K, Nakajima K, Ohta M, Okanishi T, Kanai S, Fujimoto A, Saitsu H, Matsumoto
595
N, Kato M. (2018) PLPBP mutations cause variable phenotypes of developmental
596
and epileptic encephalopathy. Epilepsia Open. 3, 495-502.
597
Takenaka T, Ito T, Miyahara I, Hemmi H, Yoshimura T. (2015) A new member of
598
MocR/GabR-type PLP-binding regulator of D-alanyl-D-alanine ligase in
599
Brevibacillus brevis. FEBS J. 282(21):4201-17.
600
I. Purification and role in pantothenate biosynthesis. J Biol Chem. 251, 3780-5.
601
602
Teller JH, Powers SG, Snell EE. (1976) Ketopantoate hydroxymethyltransferase.
Thériault O, Poulin H, Thomas GR, Friesen AD, Al-Shaqha WA, Chahine M.
603
(2014) Pyridoxal-5'-phosphate (MC-1), a vitamin B6 derivative, inhibits
604
expressed P2X receptors. Can J Physiol Pharmacol. 92(3):189-96.
24
605
Tramonti A, Nardella C, di Salvo ML, Pascarella S, Contestabile R. (2018) The
606
MocR-like transcription factors: pyridoxal 5'-phosphate-dependent regulators of
607
bacterial metabolism. FEBS J. 285, 3925-3944
608
enzymes. Biochim Biophys Acta. 1814, 1407-18.
609
610
Toney MD (2011) Controlling reaction specificity in pyridoxal phosphate
Tremiño L, Forcada-Nadal A, Contreras A, Rubio V (2017) Studies on
611
cyanobacterial protein PipY shed light on structure, potential functions, and
612
vitamin B6-dependent epilepsy. FEBS Lett 591:3431–3442.
613
Tremiño L, Forcada-Nadal A, Rubio V. (2018) Insight into vitamin B6 -dependent
614
epilepsy due to PLPBP (previously PROSC) missense mutations. Hum Mutat. 39,
615
1002-1013.
616
mediated gene expression by vitamin B6. FASEB J. 8:343-9.
617
618
Umbarger HE, Umbarger MA, Siu PM (1963) Biosynthesis of serine in
Escherichia coli and Salmonella typhimurium. J Bacteriol. 85, 1431-9.
619
620
Tully DB, Allgood VE, Cidlowski JA. (1994) Modulation of steroid receptor-
Wilson RL, Stauffer GV. (1994) DNA sequence and characterization of GcvA, a
621
LysR family regulatory protein for the Escherichia coli glycine cleavage enzyme
622
system. J Bacteriol. 176:2862-8.
623
function of xanthine dehydrogenase in purine salvage. J Bacteriol. 182, 5332-41.
624
625
Wilson MP, Plecko B, Mills PB, Clayton PT. (2019) Disorders affecting vitamin
B6 metabolism. J Inherit Metab Dis. doi: 10.1002/jimd.12060.
626
627
Xi H, Schneider BL, Reitzer L. (2000) Purine catabolism in Escherichia coli and
Zhao G, Winkler ME. (1995) Kinetic limitation and cellular amount of pyridoxine
628
(pyridoxamine) 5’-phosphate oxidase of Escherichia coli K-12. J Bacteriol.
629
177:883-91.
630
631
25
633
Table 1 Activity of GCV or GlyA in the cell-free extract of serA and serA yggS
strains
634
The serA or serA yggS strains harboring pU0 plasmid were grown in the M9-Ser or M9-
635
Gly medium. Ampicillin was added for plasmid maintenance. The E. coli strains were
636
collected at log-phase and disrupted by sonication. The enzyme activities were analyzed
637
as described in Experimental Procedures. The data represent the averages and standard
638
deviations from triplicate experiments.
632
639
640
GCV activity
GlyA activity
(5, 10-mTHF production (pmol)/mg protein)
(5, 10-mTHF production (pmol)/mg protein/min)
+ Ser
+ Gly
+ Ser
+ Gly
serA
11 ± 1
69 ± 4
170 ± 6
220 ± 12
serA yggS
66 ± 1
215 ± 19
192 ± 8
202 ± 15
641
642
26
643
Table 2 Total vitamin B6 levels in the E. coli strains
644
The serA or serA yggS strains harboring pU0 plasmid or complementary pUS plasmid
645
(expresses YggS protein) were grown in the M9-Ser or M9-Gly medium. The serA pdxH
646
strain was grown in the M9-Ser medium in the presence of 10 M PL. The glyA and glyA
647
yggS strains were grown in the M9-Casa medium. Amp (100 g/ml) was added for
648
plasmid maintenance. The E. coli strains were collected at log-phase and the total B6 pools
649
were analyzed as described in Experimental Procedures. The data represent the averages
650
and standard deviations from triplicate experiments.
651
PLP conc. (M)
PNP conc. (M)
PMP conc. (M)
(M9 + Gly)
serA
57 ± 4
7 ± 0.2
146 ± 14
serA yggS
49 ± 10
32 ± 5.8
121 ± 24
serA yggS/yggS+
62 ± 2
9 ± 0.6
147 ± 14
serA
68 ± 5
7 ± 0.4
145 ± 3
serA yggS
78 ± 4
serA yggS/yggS+
69 ± 3
serA pdxH
55.7 ± 3.6
(M9 + Ser)
38 ± 1
6 ± 0.3
132 ± 6
138 ± 6
159 ± 36
44 ± 10
(M9 + Casa)
652
glyA
83 ± 7
N.D.a
84 ± 2
glyA yggS
98 ± 11
28 ± 1
127 ± 6
, N.D.: Not Detected
653
27
654
Table 3 E. coli strains and plasmids used in this study
655
Strains
glyA
E. coli BW25113 glyA::Km (JW2535-KC)
Keio collection
glyA-Km
E. coli BW25113 glyA
This study
glyA yggS
E. coli BW25113 glyA yggS::Km
This study
serA
E. coli BW25113 serA::Km (JW2880-KC)
Keio collection
serA-Km
E. coli BW25113 serA
This study
serA yggS
E. coli BW25113 serA yggS::Km
This study
serA pdxH
E. coli BW25113 serA pdxH::Km
This study
gcvP
E. coli BW25113 gcvP::Km (JW2871-KC)
Keio collection
pU0
pUC19 containing a partial sequence of yggS
Ito et al., 2009
pUS
pUC19 expressing yggS
Ito et al., 2009
pBAD24
pBAD24 empty vector
Laboratory collection
pBAD24-pdxH
pBAD24 containing pdxH from S. enterica
Vu
Plasmids
et
al.
to
be
published
pBAD-gcvTHP pBAD-MycHisC containing gcvT-gcvH-gcvP
This study
pBAD-gcvP
pBAD-MycHisC containing gcvP
This study
pCA24N-folD
pCA24N containing folD (JW0518-AM)
ASKA clone
pKD13
A template plasmid for gene disruption. The Datsenko et al. 2000
Kmr gene is flanked by FRT sites.
pKD46
Lambda Red recombinase expression plasmid
Datsenko et al. 2000
pCP20
Yeast Flp recombinase expression plasmid with Nagarajan et al. 1997
temperature-sensitive replication.
656
28
657
Figure legends
658
Figure 1 Reaction of GlyA and GCV system
659
GlyA is PLP-dependent enzyme and catalyzes conversion of Ser to Gly, while transferring
660
the hydroxymethyl group to tetrahydrofolate (THF), and generates 5, 10-methyl-
661
tetrahydrofolate (5,10-mTHF). GCV system cleaves Gly to CO2, ammonia and provides
662
5,10-mTHF. GlyA and GCV reactions require PLP. In the wild-type E. coli, GlyA can
663
provide most of the 5,10-mTHF (Meedele et al., 1974). In the absence of glyA, GCV
664
system provides 5,10-mTHF for one-carbon biosynthesis.
665
666
Figure 2 Effect of yggS mutation under glyA background on the growths and
667
intracellular amino acid pool
668
(A) Growth of the glyA and glyA yggS mutants in the LB medium. (B) Growth of the glyA
669
strain and glyA yggS mutants both harboring pU0 plasmid (pUC19 containing partial
670
sequence of yggS) and glyA yggS mutant harboring pUS plasmid (yggS expression vector)
671
(Ito et al. 2009) in the M9-Casa medium. Cells growth was recorded by the ELx808. (C)
672
Intracellular amino acid pool of glyA or glyA yggS mutants grown in the M9-Casa medium.
673
Amino acid pools were analyzed as described in Experimental procedure. Disruption of
674
yggS under glyA background affects Ile/Val and Met metabolisms. (*p < 0.05, **p < 0.01,
675
***p <0.001, *P < 0.05, **P < 0.01, student's t-test)
676
677
Fig. 3 Effect of nucleotide and/or amino acid on the growth of glyA yggS
678
Growths of the glyA (circle) and glyA yggS double mutant (square) in the M9-Casa
679
medium in the presence of nucleotide and/or amino acid. The concentration of nucleotide
680
or amino acid (Met or Gly) was 0.2 mM or 2 mM, respectively. Guanosine (A), adenosine
681
(B), or inosine (C) supported the growth of glyA yggS double mutant. Adenine inhibited
682
the growth of the glyA (A). Other nucleotides and amino acids did not significantly affect
683
the growth of glyA strain (data not shown). The data represent the averages and standard
29
684
deviations from triplicate experiments. Cells growth was monitored by the ELx808.
685
686
Fig. 4 Effect of yggS mutation under serA background on the growths and
687
intracellular amino acid pool
688
(A, B) Growths of the serA and serA yggS double mutant in the (A) M9-Ser or (B) M9-
689
Gly medium. When grown in the M9-Ser medium, the two strains exhibited almost
690
identical growth. When grown in the M9-Gly medium, the serA yggS double mutant
691
showed poor growth. Cells growth was recorded by the ELx808. (C, D) Differences of
692
amino acid pools of serA and serA yggS grown in the (C) M9-Ser or (D) M9-Gly medium.
693
When grown in the M9-Gly medium, the amino acid pool of serA yggS strain was
694
significantly different from that of serA strain. Experiments were performed in triplicate,
695
and data are represented as the fold-change. (*p < 0.05, **p < 0.01, ***p <0.001, *P <
696
0.05, **P < 0.01, student's t-test)
697
698
Fig. 5 Effect of PN on the growths and concentrations of total B6 vitamers of serA
699
yggS strain
700
(A) Growths of the serA yggS double mutant in the M9-Ser (black) or M9-Gly medium
701
(red) in the presence (open symbol) or absence of PN (1 M) (closed symbol). When
702
grown in the M9-Gly medium, the growth of serA yggS double mutant was further
703
inhibited by PN. Cells growth was recorded by the OD-Monitor C&T apparatus. (B)
704
Concentrations of B6 vitamers in serA yggS mutant grown in the absence or presence of
705
1 M of PN. Exogenous PN significantly increased the intracellular content of PNP.
706
707
Fig. 6 PNP inhibits GCV system in vivo
708
(A, B, C, D) Growths of serA and serA yggS double mutant harboring pBAD empty vector
709
(pBAD), pBAD-pdxH, pBAD-gcvTHP (gcvTHP+), or pBAD-gcvP (gcvP+) plasmid
710
(pdxH+) were compared in the M9+Ser or M9+Gly medium. Expression of gcvT-gcvH-
30
711
gcvP (gcvTHP+), gcvP, or pdxH was induced by 0.2% or 0.02% arabinose, respectively.
712
The expression of pdxH, gcvT-gcvH-gcvP, or gcvP significantly improved the growth of
713
serA yggS double mutant in the M9-Gly medium. Note that growth of serA mutant strain
714
was not significantly affected by the overexpression of gcvP or pdxH. (E, F) Growth of
715
serA and serA pdxH double mutant in a (E) M9-Ser + 10 M PL or (F) M9-Gly + 10 M
716
PL medium. The serA pdxH double mutant exhibited lethality in the M9-Gly + 10 M PL
717
medium. Cells growth was recorded by the OD-Monitor C&T apparatus (panels C, D) or
718
the ELx808 (panels A, B, E, F) using 96-wells plate.
719
720
721
Fig. 7 Estimation of free B6 levels in the serA yggS mutant
722
The serA or serA yggS double mutant was cultivated in the M9+Gly medium. The cells
723
were disrupted and centrifuged. The resultant cell-free fraction was passed through the
724
centrifugal filter device (10 kDa-cut off) and obtained the protein-free fraction. The B6
725
levels in the cell-free fraction (total B6) or the protein-free-fraction were determined as
726
described in the Experimental procedure. In the serA yggS mutant, most of the PNP was
727
presented as free-form and the concentration was almost identical to the free PLP
728
concentration.
729
730
Fig. 8 PNP inhibits GCV system in vitro
731
GCV activity was measured in the presence of (A) 5 M or (B) 50 M of added PLP, and
732
various concentrations of PNP (0, 5, 50, or 250 M). Cell-free extract of glyA strain grown
733
in the M9-Gly medium was used for the analyses. No GCV activity was detected in the
734
absence of added PLP. (C) Effect of PNP on GlyA activity was also assayed using a
735
purified GlyA in the presence of 5 M PLP and various concentration of PNP. (D) Effect
736
of PNP on GCV activity of B. subtilis was also assayed using cell-free extract in the
737
presence of 5 M PLP and various concentration of PNP. Experiments were performed
31
738
in triplicate, and data represent the averages and standard deviations of the means.
739
740
Fig. 9 Connection of PNP, GCV system, and phenotypes observed in the yggS-
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deficient E. coli.
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Deletion of yggS in E. coli induces accumulation of PNP by unidentified mechanism.
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High-levels of PNP compete with PLP and inhibit GCV system. Disruption of GCV
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system can decrease 5,10-mTHF supply, which may decrease flux into pantothenate
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production and increase flux for Val production. Val stimulates threonine dehydratase
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(IlvA) to produce more 2-ketobutyrate (2-KB) as a precursor for 2-aminobutyrate (2-AB),
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Ile, and ophthalmic acid (OA).
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Acknowledgment
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This work was supported by grants from the JSPS KAKENHI (grants 16K18686 and
17KK0153 to T.I.), and competitive grant GM095837 from the National Institutes of
Health (to DMD). The funders had no role in study design, data collection, and
interpretation, or the decision to submit the work for publication. No conflict of interest
is declared.
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