PHLD_PSEF5
ID PHLD_PSEF5 Reviewed; 349 AA.
AC Q4K418;
DT 22-APR-2020, integrated into UniProtKB/Swiss-Prot.
DT 02-AUG-2005, sequence version 1.
DT 03-AUG-2022, entry version 88.
DE RecName: Full=Phloroglucinol synthase {ECO:0000303|PubMed:16931521};
DE EC=2.3.1.253 {ECO:0000269|PubMed:15826166, ECO:0000269|PubMed:16931521, ECO:0000269|PubMed:18437267};
DE AltName: Full=Type III polyketide synthase PhlD {ECO:0000305};
GN Name=phlD {ECO:0000303|PubMed:15826166};
GN OrderedLocusNames=PFL_5957 {ECO:0000312|EMBL:AAY95147.1};
OS Pseudomonas fluorescens (strain ATCC BAA-477 / NRRL B-23932 / Pf-5).
OC Bacteria; Proteobacteria; Gammaproteobacteria; Pseudomonadales;
OC Pseudomonadaceae; Pseudomonas.
OX NCBI_TaxID=220664;
RN [1]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=ATCC BAA-477 / NRRL B-23932 / Pf-5;
RX PubMed=15980861; DOI=10.1038/nbt1110;
RA Paulsen I.T., Press C.M., Ravel J., Kobayashi D.Y., Myers G.S.A.,
RA Mavrodi D.V., DeBoy R.T., Seshadri R., Ren Q., Madupu R., Dodson R.J.,
RA Durkin A.S., Brinkac L.M., Daugherty S.C., Sullivan S.A., Rosovitz M.J.,
RA Gwinn M.L., Zhou L., Schneider D.J., Cartinhour S.W., Nelson W.C.,
RA Weidman J., Watkins K., Tran K., Khouri H., Pierson E.A., Pierson L.S. III,
RA Thomashow L.S., Loper J.E.;
RT "Complete genome sequence of the plant commensal Pseudomonas fluorescens
RT Pf-5.";
RL Nat. Biotechnol. 23:873-878(2005).
RN [2]
RP FUNCTION, CATALYTIC ACTIVITY, AND BIOPHYSICOCHEMICAL PROPERTIES.
RX PubMed=15826166; DOI=10.1021/ja042340g;
RA Achkar J., Xian M., Zhao H., Frost J.W.;
RT "Biosynthesis of phloroglucinol.";
RL J. Am. Chem. Soc. 127:5332-5333(2005).
RN [3]
RP FUNCTION, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, AND
RP MUTAGENESIS OF MET-21; HIS-24; LEU-59; ALA-60 AND ALA-185.
RC STRAIN=ATCC BAA-477 / NRRL B-23932 / Pf-5;
RX PubMed=16931521; DOI=10.1074/jbc.m606500200;
RA Zha W., Rubin-Pitel S.B., Zhao H.;
RT "Characterization of the substrate specificity of PhlD, a type III
RT polyketide synthase from Pseudomonas fluorescens.";
RL J. Biol. Chem. 281:32036-32047(2006).
RN [4]
RP CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, BIOTECHNOLOGY, AND
RP MUTAGENESIS OF LYS-210; TYR-256 AND ALA-289.
RC STRAIN=ATCC BAA-477 / NRRL B-23932 / Pf-5;
RX PubMed=18437267; DOI=10.1039/b717705d;
RA Zha W., Rubin-Pitel S.B., Zhao H.;
RT "Exploiting genetic diversity by directed evolution: molecular breeding of
RT type III polyketide synthases improves productivity.";
RL Mol. Biosyst. 4:246-248(2008).
RN [5]
RP BIOTECHNOLOGY.
RC STRAIN=ATCC BAA-477 / NRRL B-23932 / Pf-5;
RX PubMed=21643705; DOI=10.1007/s00253-011-3304-5;
RA Cao Y., Jiang X., Zhang R., Xian M.;
RT "Improved phloroglucinol production by metabolically engineered Escherichia
RT coli.";
RL Appl. Microbiol. Biotechnol. 91:1545-1552(2011).
RN [6]
RP BIOTECHNOLOGY, AND MUTAGENESIS OF MET-21; ASN-27; LEU-54; ALA-82; SER-96
RP AND ALA-181.
RX PubMed=23358999; DOI=10.1007/s00253-013-4713-4;
RA Rao G., Lee J.K., Zhao H.;
RT "Directed evolution of phloroglucinol synthase PhlD with increased
RT stability for phloroglucinol production.";
RL Appl. Microbiol. Biotechnol. 97:5861-5867(2013).
CC -!- FUNCTION: Type III polyketide synthase that catalyzes the synthesis of
CC phloroglucinol from three molecules of malonyl-CoA (PubMed:15826166,
CC PubMed:16931521). In addition to its ability to produce phloroglucinol
CC from malonyl-CoA, it exhibits broad substrate specificity, accepting
CC C4-C12 aliphatic acyl-CoAs and phenylacetyl-CoA as the starters to form
CC C6-polyoxoalkylated alpha-pyrones from sequential condensation with
CC malonyl-CoA (PubMed:16931521). {ECO:0000269|PubMed:15826166,
CC ECO:0000269|PubMed:16931521}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=3 H(+) + 3 malonyl-CoA = 1,3,5-trihydroxybenzene + 3 CO2 + 3
CC CoA; Xref=Rhea:RHEA:50380, ChEBI:CHEBI:15378, ChEBI:CHEBI:16204,
CC ChEBI:CHEBI:16526, ChEBI:CHEBI:57287, ChEBI:CHEBI:57384;
CC EC=2.3.1.253; Evidence={ECO:0000269|PubMed:15826166,
CC ECO:0000269|PubMed:16931521, ECO:0000269|PubMed:18437267};
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Kinetic parameters:
CC KM=5.6 uM for malonyl-CoA {ECO:0000269|PubMed:15826166};
CC KM=13 uM for malonyl-CoA {ECO:0000269|PubMed:16931521};
CC KM=13.1 uM for malonyl-CoA {ECO:0000269|PubMed:18437267};
CC Note=kcat is 10 min(-1) with malonyl-CoA as substrate
CC (PubMed:15826166). kcat is 24 min(-1) with malonyl-CoA as substrate
CC (PubMed:16931521). kcat is 7.3 min(-1) with malonyl-CoA as substrate
CC (PubMed:18437267). {ECO:0000269|PubMed:15826166,
CC ECO:0000269|PubMed:16931521, ECO:0000269|PubMed:18437267};
CC -!- PATHWAY: Antibiotic biosynthesis. {ECO:0000250|UniProtKB:Q51725}.
CC -!- BIOTECHNOLOGY: Phloroglucinol is widely used in industry and in the
CC synthesis of pharmaceuticals. PhlD can be used to produce this
CC chemical, however it exhibits low productivity, which is a bottleneck
CC for large-scale application. Different stategies have been developed to
CC improve the stability and the productivity of the enzyme.
CC {ECO:0000269|PubMed:18437267, ECO:0000269|PubMed:21643705,
CC ECO:0000269|PubMed:23358999}.
CC -!- SIMILARITY: Belongs to the thiolase-like superfamily. Chalcone/stilbene
CC synthases family. {ECO:0000305}.
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DR EMBL; CP000076; AAY95147.1; -; Genomic_DNA.
DR RefSeq; WP_011064130.1; NC_004129.6.
DR AlphaFoldDB; Q4K418; -.
DR SMR; Q4K418; -.
DR STRING; 220664.PFL_5957; -.
DR EnsemblBacteria; AAY95147; AAY95147; PFL_5957.
DR GeneID; 57478912; -.
DR KEGG; pfl:PFL_5957; -.
DR PATRIC; fig|220664.5.peg.6073; -.
DR eggNOG; COG3424; Bacteria.
DR HOGENOM; CLU_034992_0_0_6; -.
DR OMA; PIWGLGC; -.
DR OrthoDB; 872193at2; -.
DR BioCyc; MetaCyc:MON-19911; -.
DR BRENDA; 2.3.1.253; 5121.
DR Proteomes; UP000008540; Chromosome.
DR GO; GO:0034083; C:type III polyketide synthase complex; IDA:JCVI.
DR GO; GO:0016747; F:acyltransferase activity, transferring groups other than amino-acyl groups; IEA:InterPro.
DR GO; GO:0017000; P:antibiotic biosynthetic process; IEA:UniProtKB-KW.
DR GO; GO:0030639; P:polyketide biosynthetic process; IDA:JCVI.
DR Gene3D; 3.40.47.10; -; 2.
DR InterPro; IPR012328; Chalcone/stilbene_synt_C.
DR InterPro; IPR001099; Chalcone/stilbene_synt_N.
DR InterPro; IPR011141; Polyketide_synthase_type-III.
DR InterPro; IPR016039; Thiolase-like.
DR PANTHER; PTHR11877; PTHR11877; 1.
DR Pfam; PF02797; Chal_sti_synt_C; 1.
DR Pfam; PF00195; Chal_sti_synt_N; 1.
DR PIRSF; PIRSF000451; PKS_III; 1.
DR SUPFAM; SSF53901; SSF53901; 2.
PE 1: Evidence at protein level;
KW Acyltransferase; Antibiotic biosynthesis; Reference proteome; Transferase.
FT CHAIN 1..349
FT /note="Phloroglucinol synthase"
FT /id="PRO_0000449605"
FT ACT_SITE 138
FT /evidence="ECO:0000250|UniProtKB:Q54240"
FT MUTAGEN 21
FT /note="M->I: Does not affect phloroglucinol production but
FT shows 50% decrease in activity toward lauroyl-CoA."
FT /evidence="ECO:0000269|PubMed:16931521"
FT MUTAGEN 21
FT /note="M->T: No change in activity toward lauroyl-CoA. 3.8-
FT fold improvement in thermostability. Shows 90% of wild type
FT specific activity."
FT /evidence="ECO:0000269|PubMed:16931521,
FT ECO:0000269|PubMed:23358999"
FT MUTAGEN 24
FT /note="H->L: Shows reduced activity toward lauroyl-CoA and
FT decrease in phloroglucinol production."
FT /evidence="ECO:0000269|PubMed:16931521"
FT MUTAGEN 24
FT /note="H->V: Does not affect phloroglucinol production but
FT shows 90% decrease in activity toward lauroyl-CoA."
FT /evidence="ECO:0000269|PubMed:16931521"
FT MUTAGEN 27
FT /note="N->D: 6.5-fold improvement in thermostability. Shows
FT 90% of wild type specific activity."
FT /evidence="ECO:0000269|PubMed:23358999"
FT MUTAGEN 54
FT /note="L->V: 1.9-fold improvement in thermostability. Shows
FT 110% of wild type specific activity."
FT /evidence="ECO:0000269|PubMed:23358999"
FT MUTAGEN 59
FT /note="L->M: Does not affect phloroglucinol production but
FT shows 50% decrease in activity toward lauroyl-CoA."
FT /evidence="ECO:0000269|PubMed:16931521"
FT MUTAGEN 60
FT /note="A->L: No change in activity toward lauroyl-CoA."
FT /evidence="ECO:0000269|PubMed:16931521"
FT MUTAGEN 82
FT /note="A->T: 3.8-fold improvement in thermostability. Shows
FT wild type specific activity."
FT /evidence="ECO:0000269|PubMed:23358999"
FT MUTAGEN 96
FT /note="S->R: 2.3-fold improvement in thermostability. Shows
FT 80% of wild type specific activity."
FT /evidence="ECO:0000269|PubMed:23358999"
FT MUTAGEN 181
FT /note="A->S: 2.5-fold improvement in thermostability. Shows
FT 110% of wild type specific activity."
FT /evidence="ECO:0000269|PubMed:23358999"
FT MUTAGEN 185
FT /note="A->D: Loss of activity."
FT /evidence="ECO:0000269|PubMed:16931521"
FT MUTAGEN 210
FT /note="K->L: 2-fold increase in catalytic efficiency."
FT /evidence="ECO:0000269|PubMed:18437267"
FT MUTAGEN 256
FT /note="Y->R: 2-fold increase in catalytic efficiency."
FT /evidence="ECO:0000269|PubMed:18437267"
FT MUTAGEN 289
FT /note="A->R: 1.5-fold increase in catalytic efficiency."
FT /evidence="ECO:0000269|PubMed:18437267"
SQ SEQUENCE 349 AA; 38617 MW; FBBC903E7F3AE4F7 CRC64;
MSTLCLPHVM FPQHKITQQQ MVDHLENLHA DHPRMALAKR MIANTEVNER HLVLPIDELA
VHTGFTHRSI VYEREARQMS SAAARQAIEN AGLQISDIRM VIVTSCTGFM MPSLTAHLIN
DLALPTSTVQ LPIAQLGCVA GAAAINRAND FARLDARNHV LIVSLEFSSL CYQPDDTKLH
AFISAALFGD AVSACVLRAD DQAGGFKIKK TESYFLPKSE HYIKYDVKDT GFHFTLDKAV
MNSIKDVAPV MERLNYESFE QNCAHNDFFI FHTGGRKILD ELVMHLDLAS NRVSQSRSSL
SEAGNIASVV VFDVLKRQFD SNLNRGDIGL LAAFGPGFTA EMAVGEWTA
