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Escherichia coli K-12 substr. MG1655 Enzyme: pyridoxine 5'-phosphate oxidase / pyridoxamine 5'-phosphate oxidase



Gene: pdxH Accession Numbers: EG11487 (EcoCyc), b1638, ECK1634

Regulation Summary Diagram: ?

Subunit composition of pyridoxine 5'-phosphate oxidase / pyridoxamine 5'-phosphate oxidase = [PdxH]2

Summary:
Pyridoxine 5'-phosphate oxidase encoded by the pdxH gene catalyzes the oxidation of pyridoxine 5'-phosphate (PNP) to Pyridoxal 5'-phosphate (PLP) in the presence and absence of molecular oxygen in the final step of vitamin B6 biosynthesis in E. coli. These growth properties could be explained either by the presence of a second enzyme that replaces the oxidase function, which might still require the pdxH gene product for function / expression or by the ability of PNPOx to function with a final electron acceptor different from oxygen. The enzyme catalyzes the oxidation of either the C4' alcohol group or amino group of the two substrates pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) to an aldehyde, forming PLP. The loss of the enzyme activity disrupts the cellular metabolism. In E. coli it results in excretion of L-glutamate and possibly α-ketoisovalerate, which triggers L-valine inhibition [diSalvo03].

The enzyme is relatively abundant, but catalytically sluggish. PNP rather than PMP is the in vivo substrate of E. coli. The reaction catalyzed by the enzyme serves as a critical constituent in the recycling of PLP. There might be a second pathway for PLP biosynthesis in anaerobically growing E. coli cells [Zhao95a].

PLP molecules bind tightly to both apo and holo enzyme with a stoichiometry of one PLP per monomer. Holo-enzyme with tightly bound PLP shows normal catalytic activity, suggesting that the tightly bound PLP is at a noncatalytic site. The enzyme targets PLP to apo serine hydroxymethyltransferase in vivo [Yang00a]. A tunnel exists between the two sites of the enzyme so that pyridoxal 5'-phosphate formed at the active site may transfer to the non-catalytic site without passing though the solvent. Asn84, Phe177 and Lys145 are critical in binding PLP at the noncatalytic site [Safo01].

The enzyme is a homodimer with two molecules of flavin mononucleotide (FMN) per dimer [Di98]. The crystal structure shows that one FMN molecule is covalently bound to each subunit. FMN molecules are involved in extensive hydrophobic and hydrogen bond interactions with the oxidase dimer, contributing to the contact surface area. Tyr17 and Arg14 are a part of the N-terminal region that covers the active site [Safo00].

Each subunit of the enzyme consists of two domains. A crystal structure in a tetragonal unit cell shows major disorder in most of the smaller domain 2 residues that has opened up both the active site and a putative tunnel. This disorder suggests that domain 2 could easily rotate to allow passage of PLP from the active site to the putative non-catalytic secondary PLP site [Safo05].

The orientation of the bound pyridoxal 5'-phosphate suggests that the enzyme catalyzes a hydride ion transfer between C4' of pyridoxal 5'-phosphate and N5 of FMN [Safo01].

Gene Citations: [Yang98b]

Locations: cytosol

Map Position: [1,715,375 <- 1,716,031] (36.97 centisomes)
Length: 657 bp / 218 aa

Molecular Weight of Polypeptide: 25.545 kD (from nucleotide sequence)

pI: 9.3

Unification Links: ASAP:ABE-0005482 , CGSC:417 , DIP:DIP-48024N , DisProt:DP00165 , EchoBASE:EB1450 , EcoGene:EG11487 , EcoliWiki:b1638 , Entrez-gene:946806 , ModBase:P0AFI7 , OU-Microarray:b1638 , PortEco:pdxH , PR:PRO_000023504 , Pride:P0AFI7 , Protein Model Portal:P0AFI7 , RefSeq:NP_416155 , RegulonDB:EG11487 , SMR:P0AFI7 , String:511145.b1638 , Swiss-Model:P0AFI7 , UniProt:P0AFI7

Relationship Links: InterPro:IN-FAMILY:IPR000659 , InterPro:IN-FAMILY:IPR011576 , InterPro:IN-FAMILY:IPR012349 , InterPro:IN-FAMILY:IPR019576 , InterPro:IN-FAMILY:IPR019740 , Panther:IN-FAMILY:PTHR10851 , PDB:Structure:1DNL , PDB:Structure:1G76 , PDB:Structure:1G77 , PDB:Structure:1G78 , PDB:Structure:1G79 , PDB:Structure:1JNW , PDB:Structure:1WV4 , Pfam:IN-FAMILY:PF01243 , Pfam:IN-FAMILY:PF10590 , Prosite:IN-FAMILY:PS01064

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0008615 - pyridoxine biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA11, GOA01, diSalvo03, Zhao95a]
GO:0009443 - pyridoxal 5'-phosphate salvage Inferred from experiment [diSalvo03]
GO:0055114 - oxidation-reduction process Inferred from experiment Inferred by computational analysis [UniProtGOA11, GOA01, Zhao95a]
GO:0042816 - vitamin B6 metabolic process Inferred by computational analysis [GOA06]
Molecular Function: GO:0004733 - pyridoxamine-phosphate oxidase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, GOA01, Zhao95a]
GO:0010181 - FMN binding Inferred from experiment Inferred by computational analysis [GOA06, GOA01, diSalvo03, Zhao95a]
GO:0016491 - oxidoreductase activity Inferred from experiment Inferred by computational analysis [UniProtGOA11, GOA01, Zhao95a]
GO:0016638 - oxidoreductase activity, acting on the CH-NH2 group of donors Inferred by computational analysis [GOA01]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]

MultiFun Terms: metabolism biosynthesis of building blocks cofactors, small molecule carriers pyridoxal 5'phosphate
metabolism central intermediary metabolism pyridoxal 5'-phosphate salvage

Essentiality data for pdxH knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 0.4% glucose No 37 Aerobic 7.2 0.27 No [Patrick07, Comment 3]
M9 medium with 1% glycerol No 37 Aerobic 7.2 0.35 No [Joyce06]
MOPS medium with 0.4% glucose Indeterminate 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
No [Feist07, Comment 4]

Credits:
Reviewed 19-Mar-2010 by Sarker M
Last-Curated ? 13-Aug-2013 by Kubo A , SRI International


Enzymatic reaction of: pyridoxine 5'-phosphate oxidase

Synonyms: PNP oxidase, pyridoxamine-phosphate oxidase, pyridoxamine-5'-phosphate oxidase, PMP oxidase, pyridoxamine-5'-phosphate:oxygen oxidoreductase (deaminating)

EC Number: 1.4.3.5

pyridoxine 5'-phosphate + oxygen <=> hydrogen peroxide + pyridoxal 5'-phosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

The reaction is physiologically favored in the direction shown.

In Pathways: superpathway of pyridoxal 5'-phosphate biosynthesis and salvage , pyridoxal 5'-phosphate biosynthesis I , pyridoxal 5'-phosphate salvage I

Summary:
Molecular oxygen is the ultimate electron acceptor of the enzymatic reaction, producing hydrogen peroxide.

Substrate inhibition occurs by binding of PNP to the reduced form of the enzyme, forming an abortive complex.

The enzyme activity are competitively inhibited by PLP, and by the analog, 4-deoxy-PNP, with Ki values of 8 and 105 uM, respectively. PLP and PLP analogs inactivate the enzyme by binding to a specific lysine residue in active site of the dimeric form of the protein. Since PLP is a very reactive molecule that forms complexes with most small nucleophiles and proteins in the cell, the pool of free PLP is maintained at a very low level. Product inhibition by PLP is one such mechanism for regulation [Zhao95a].

Cofactors or Prosthetic Groups: FMN [Comment 5, Zhao95a]

Inhibitors (Competitive): 4-deoxypyridoxine 5'-phosphate [Zhao95a] , pyridoxal 5'-phosphate [Zhao95a, Comment 6]

Inhibitors (Unknown Mechanism): pyridoxine 5'-phosphate [Zhao95a, Comment 7]

Kinetic Parameters:

Substrate
Km (μM)
Citations
pyridoxine 5'-phosphate
2.0
[Zhao95a]

T(opt): 50 °C [Zhao95a]

pH(opt): 8.5 [Zhao95a]


Enzymatic reaction of: pyridoxamine 5'-phosphate oxidase

Synonyms: PNP oxidase, pyridoxamine-phosphate oxidase, pyridoxamine-5'-phosphate oxidase, PMP oxidase, pyridoxamine-5'-phosphate:oxygen oxidoreductase (deaminating)

EC Number: 1.4.3.5

pyridoxamine 5'-phosphate + oxygen + H2O <=> ammonium + hydrogen peroxide + pyridoxal 5'-phosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

The reaction is physiologically favored in the direction shown.

Alternative Substrates [Comment 8]:

In Pathways: superpathway of pyridoxal 5'-phosphate biosynthesis and salvage , pyridoxal 5'-phosphate salvage I

Cofactors or Prosthetic Groups: FMN [Comment 5]

Inhibitors (Competitive): 4-deoxypyridoxine 5'-phosphate [Zhao95a] , pyridoxal 5'-phosphate [Comment 6]


Sequence Features

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
[Zhao95a, UniProt11]
UniProt: Removed.
Chain 2 -> 218
[UniProt09]
UniProt: Pyridoxine/pyridoxamine 5'-phosphate oxidase;
Mutagenesis-Variant 14
[diSalvo02, UniProt11]
Alternate sequence: R → M; UniProt: Reduces affinity for substrate about 9-fold, but has no effect on catalytic activity.
Alternate sequence: R → E; UniProt: Reduces affinity for substrate about 7-fold, but has no effect on catalytic activity.
Protein-Segment 14 -> 17
[UniProt10a]
UniProt: Substrate binding; Sequence Annotation Type: region of interest;
Mutagenesis-Variant 17
[diSalvo02, UniProt11]
Alternate sequence: Y → F; UniProt: Reduces affinity for substrate 3- fold, but has about 5-fold increase in catalytic activity.
Mutagenesis-Variant 49
[diSalvo02, UniProt11]
Alternate sequence: D → A; UniProt: Reduces affinity for substrate 3- fold and catalytic activity 2-fold.
Amino-Acid-Sites-That-Bind 67
[UniProt10]
UniProt: FMN;
Amino-Acid-Sites-That-Bind 70
[UniProt10]
UniProt: FMN; via amide nitrogen;
Amino-Acid-Sites-That-Bind 72
[UniProt10]
UniProt: Substrate;
Nucleotide-Phosphate-Binding-Region 82 -> 83
[UniProt10]
UniProt: FMN;
Amino-Acid-Sites-That-Bind 89
[UniProt10]
UniProt: FMN;
Amino-Acid-Sites-That-Bind 129
[UniProt10]
UniProt: Substrate;
Amino-Acid-Sites-That-Bind 133
[UniProt10]
UniProt: Substrate;
Amino-Acid-Sites-That-Bind 137
[UniProt10]
UniProt: Substrate;
Nucleotide-Phosphate-Binding-Region 146 -> 147
[UniProt10]
UniProt: FMN;
Mutagenesis-Variant 197
[diSalvo02, UniProt11]
Alternate sequence: R → M; UniProt: Reduces affinity for substrate 300- fold and catalytic activity about 4-fold.
Alternate sequence: R → E; UniProt: Reduces affinity for substrate 8000-fold and catalytic activity 16-fold.
Protein-Segment 197 -> 199
[UniProt10a]
UniProt: Substrate binding; Sequence Annotation Type: region of interest;
Mutagenesis-Variant 199
[diSalvo02, UniProt11]
Alternate sequence: H → N; UniProt: Reduces catalytic activity about 4- fold, but has no effect on affinity for substrate.
Alternate sequence: H → A; UniProt: Reduces affinity for substrate 230- fold, but has no effect on catalytic activity.


Gene Local Context (not to scale): ?

Transcription Units:

Notes:

History:
10/20/97 Gene b1638 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11487; confirmed by SwissProt match.


References

Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

Di98: Di Salvo M, Yang E, Zhao G, Winkler ME, Schirch V (1998). "Expression, purification, and characterization of recombinant Escherichia coli pyridoxine 5'-phosphate oxidase." Protein Expr Purif 1998;13(3);349-56. PMID: 9693059

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

diSalvo02: di Salvo ML, Ko TP, Musayev FN, Raboni S, Schirch V, Safo MK (2002). "Active site structure and stereospecificity of Escherichia coli pyridoxine-5'-phosphate oxidase." J Mol Biol 315(3);385-97. PMID: 11786019

diSalvo03: di Salvo ML, Safo MK, Musayev FN, Bossa F, Schirch V (2003). "Structure and mechanism of Escherichia coli pyridoxine 5'-phosphate oxidase." Biochim Biophys Acta 1647(1-2);76-82. PMID: 12686112

Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909

Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA01a: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Ishihama08: Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D (2008). "Protein abundance profiling of the Escherichia coli cytosol." BMC Genomics 9;102. PMID: 18304323

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

Patrick07: Patrick WM, Quandt EM, Swartzlander DB, Matsumura I (2007). "Multicopy suppression underpins metabolic evolvability." Mol Biol Evol 24(12);2716-22. PMID: 17884825

Safo00: Safo MK, Mathews I, Musayev FN, di Salvo ML, Thiel DJ, Abraham DJ, Schirch V (2000). "X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with FMN at 1.8 A resolution." Structure 8(7);751-62. PMID: 10903950

Safo01: Safo MK, Musayev FN, di Salvo ML, Schirch V (2001). "X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with pyridoxal 5'-phosphate at 2.0 A resolution." J Mol Biol 310(4);817-26. PMID: 11453690

Safo05: Safo MK, Musayev FN, Schirch V (2005). "Structure of Escherichia coli pyridoxine 5'-phosphate oxidase in a tetragonal crystal form: insights into the mechanistic pathway of the enzyme." Acta Crystallogr D Biol Crystallogr 61(Pt 5);599-604. PMID: 15858270

UniProt09: UniProt Consortium (2009). "UniProt version 15.8 released on 2009-10-01 00:00:00." Database.

UniProt10: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt11: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

Yang00a: Yang ES, Schirch V (2000). "Tight binding of pyridoxal 5'-phosphate to recombinant Escherichia coli pyridoxine 5'-phosphate oxidase." Arch Biochem Biophys 377(1);109-14. PMID: 10775448

Yang98b: Yang Y, Tsui HC, Man TK, Winkler ME (1998). "Identification and function of the pdxY gene, which encodes a novel pyridoxal kinase involved in the salvage pathway of pyridoxal 5'-phosphate biosynthesis in Escherichia coli K-12." J Bacteriol 1998;180(7);1814-21. PMID: 9537380

Zhao95a: Zhao G, Winkler ME (1995). "Kinetic limitation and cellular amount of pyridoxine (pyridoxamine) 5'-phosphate oxidase of Escherichia coli K-12." J Bacteriol 1995;177(4);883-91. PMID: 7860596

Other References Related to Gene Regulation

Lam92: Lam HM, Winkler ME (1992). "Characterization of the complex pdxH-tyrS operon of Escherichia coli K-12 and pleiotropic phenotypes caused by pdxH insertion mutations." J Bacteriol 174(19);6033-45. PMID: 1356963

Zaslaver06: Zaslaver A, Bren A, Ronen M, Itzkovitz S, Kikoin I, Shavit S, Liebermeister W, Surette MG, Alon U (2006). "A comprehensive library of fluorescent transcriptional reporters for Escherichia coli." Nat Methods 3(8);623-8. PMID: 16862137


Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
Page generated by SRI International Pathway Tools version 18.5 on Sun Nov 23, 2014, biocyc13.