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2.5.1.61: hydroxymethylbilane synthase

This is an abbreviated version!
For detailed information about hydroxymethylbilane synthase, go to the full flat file.

Word Map on EC 2.5.1.61

Reaction

4 porphobilinogen +

H2O
=
Hydroxymethylbilane
 + 4 NH3

Synonyms

(HMB)-synthase, AN0121.3, EC 4.3.1.8, HemC, hepatic porphobilinogen deaminase, HMB synthase, HMB-S, HMBS, HMBS1a-synthase, HMBS1b-synthase, HMBS2a-synthase, HMBS2b-synthase, human non-erythropoietic PBGD isoform, human PBGD, human porphobilinogen deaminase, hydroxymethylbilane synthase, More, PBG deaminase, PBG-D, PBG-deaminase, PBGD, PGB-D, porphobilinogen ammonia-lyase (polymerizing), porphobilinogen deaminase, pre-uroporphyrinogen synthase, preuroporphyrinogen synthase, preuroporphyrinogen synthetase, synthase, uroporphyrinogen I, uPBGD, UPGI-S, URO-S, urogenI synthase, uroporphyrinogen I synthase, uroporphyrinogen I synthetase, uroporphyrinogen synthase, uroporphyrinogen synthetase

ECTree

     2 Transferases
         2.5 Transferring alkyl or aryl groups, other than methyl groups
             2.5.1 Transferring alkyl or aryl groups, other than methyl groups (only sub-subclass identified to date)
                2.5.1.61 hydroxymethylbilane synthase

Purification

Purification on EC 2.5.1.61 - hydroxymethylbilane synthase

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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
2 forms
-
3 isoenzymes
-
5 forms, A: native enzyme, B-E: isomeres corresponding to the enzyme-substrate intermediates
-
blood samples are washed in phosphate buffered saline, liver rinsed in phosphate buffered saline
cell harvesting by centrifugation, washed, resuspended in NaCl-phosphate buffer containing of 140 mN NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3 with protease inhibitor cocktail, and 0.5% Triton X-100, lysis by shaking with lysozyme on ice, sonication, centrifugation, supernatant loaded onto glutathione sepharose 4B column, washed with 20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40+, pH 8.2, proteins eluted in 50 mM Tris-HCl, pH 8.3 buffer with 20 mM glutathione, thrombin digestion with thrombin at 20 U/mg protein at 20°C overnight, addition of glycerol to a concentration of 20%
cells centrifuged, washed with 0.9 M NaCl, re-centrifuged, washed with 20 mM Tris-HCl buffer, pH 8.2, with 5 mM dithiothreitol, and 200 microM PMSF, sonication, heating to 60°C under N2 gas, cooled to 4°C, supernatant applied to a Pharmacia 50 K/30 column packed with DEAE-Sephacel anion-exchange resin, equilibrated with 50 mM Tris-HCl buffer, pH 8.2 containing 5 mM dithiothreitol, and 100 microM PMSF, elution with 0-70 mM KCl gradient, active fractions are pooled and concentrated by ultrafiltration with a PM-10 membrane, further concentrated with a Centricon YM-10 centrifugal filter and gel-filtered on a Hiload 16/60 Superdex G-75 column, equilibrated with 100 mM Tris-HCl buffer, pH 8.2, containing 5 mM dithiothreitol and 100 microM PMSF, concentrated with a Centricon YM-10, buffer-exchanged into 20 mM Tris-HCl buffer, pH 8.2, containing 5 mM dithiothreitol with a Pharmacia PD10 column
cells harvested and resuspended in 20 mM Tris-HCl buffer, pH 8.0 containing 500 mM NaCl and 10 mM imidazole, cells are lysed by sonication and centrifuged, applied to NiCl2 Sepahrose resin and eluted with buffer and 500 mM imidazole, pooled, concentrated by ultra centrifugation (10 kDa), buffer exchanged with 50 mM Tris-HCl, pH 8.0, with a PD10 column
-
dye-ligand affinity chromatography
-
glutathione Sepharose 4B column chromatography
-
Hi-Trap chelating metal affinity column chromatography
leaves of 8 week old field grown plants are ground in liquid nitrogen and extracted with 25 mM NaP buffer, pH 8.0, containing 1 mM EDTA, 5 mM DTT, and 0.5 mM phenylmethanesulfonylfluoride, centrifuged, supernatant taken for enzyme assay
-
Ni2+-NTA resin chromatography
packed red blood cells are washed in phosphate buffered saline, lysed with buffer containing 0.1 M KHEPES, pH 7.5, 0.1% Triton X-100, 1 mM DTT, and 0.02% sodium azide, liver and spleen are diced and homogenized in buffer containing 50 mM KHEPES, pH 7.5, 150 mM KCl, 1 mM Na2EDTA, 1 mM DTT, 0.1 mM PMSF, for activity and thermostability experiments enzymes are expressed in Escherichia coli, cells are harvested and resuspended in 50 mM HEPES, pH 7.8, 0.5 M NaCl, 5 mM DTT, 330 microg/ml lysozyme, 5 microg/ml DNAse and RNAse, freeze-thawed 3times in dry-ice/ethanol, centrifuged, adjusted to pH 7.4 and 8.0 with NaOH, incubated at 37°C or 50°C for 1 h
partial
recombinant enzyme from Escherichia coli by ion exchange chromatography and gel filtration
recombinant GST-tagged isoallelic forms K210 and E210 mutants from Escherichia coli to homogeneity
-
recombinant GST-tagged wild-type and mutant enzymes from Escherichia coli BL21 (DE3) by glutathione affinity chromatography, the GST-tag is cleaved off
-
recombinant GST-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) pLysS by glutathione affinity chromatgraphy, tag cleavage by thrombin, and ultrafiltration
recombinant His-tagged enzyme from Escherichia coli strain Rosetta (DE3) by nickel affinity chromatography, removal of the His-tag by thrombin and tag elimination by another step of nickel affinity chromatography
recombinant His-tagged enzyme from Escherichia coli, removal of the His-tag
SeMet-labelled enzyme, i.e. [SeMet] HMBS, and wild-type enzyme
-