Polyhydroxyalkanoate (PHA) biosynthesis in Thermus thermophilus: purification and biochemical properties of PHA synthase.
The biosynthesis of polyhydroxyalkanoates (PHAs) was studied, for the first time, in the thermophilic bacterium Thermus thermophilus. Using sodium gluconate (1.5% w/v) or sodium octanoate (10 mM) as sole carbon sources, PHAs were accumulated to approximately 35 or 40% of the cellular dry weight, respectively. Gas chromatographic analysis of PHA isolated from gluconate-grown cells showed that the polyester (Mw: 480,000 g mol(-1)) was mainly composed of 3-hydroxydecanoate (3HD) with a molar fraction of 64%. In addition, 3-hydroxyoctanoate (3HO), 3-hydroxyvalerate (3HV) and 3-hydroxybutyrate (3HB) occurred as constituents. In contrast, the polyester (Mw: 391,000 g mol(-1)) from octanoate-grown cells was composed of 24.5 mol% 3HB, 5.4 mol% 3HO, 12.3 mol% 3-hydroxynonanoate (3HN), 14.6 mol% 3HD, 35.4 mol% 3-hydroxyundecanoate (3HUD) and 7.8 mol% 3-hydroxydodecanoate (3HDD). Activities of PHA synthase, a beta-ketothiolase and an NADPH-dependent reductase were detected in the soluble cytosolic fraction obtained from gluconate-grown cells of T. thermophilus. The soluble PHA synthase was purified 4271-fold with 8.5% recovery from gluconate-grown cells, presenting a Km of 0.25 mM for 3HB-CoA. The optimal temperature of PHA synthase activity was about 70 degrees C and acts optimally at pH near 7.3. PHA synthase activity was inhibited 50% with 25 microM CoA and lost all of its activity when it was treated with alkaline phosphatase. T. thermophilus PHA synthase, in contrary to other reported PHA synthases did not exhibit a lag phase on its kinetics, when low concentration of the enzyme was used. Incubation of PHA synthase with 1 mM N-ethyl-maleimide inhibits the enzyme 56%, indicating that cysteine might be involved in the catalytic site of the enzyme. Acetyl phosphate (10 mM) activated both the native and the dephosphorylated enzyme. A major protein (55 kDa) was detected by SDS-PAGE. When a partially purified preparation was analyzed on native PAGE the major band exhibiting PHA synthase activity was eluted from the gel and analyzed further on SDS-PAGE, presenting the first purification of a PHA synthase from a thermophilic microorganism.
Pantazaki
Anastasia A
AA
Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece. natasa@chem.auth.gr
Tambaka
Maria G
MG
Langlois
Valerie
V
Guerin
Philippe
P
Kyriakidis
Dimitrios A
DA
D020155:3-Hydroxybutyric Acid / Q000737:chemistry; D000102:Acetyl-CoA C-Acyltransferase / Q000737:chemistry / Q000378:metabolism; D000217:Acyltransferases / Q000737:chemistry* / Q000302:isolation & purification*; D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism; D002210:Caprylates / Q000737:chemistry / Q000494:pharmacology; D002244:Carbon / Q000737:chemistry; D002264:Carboxylic Acids / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D002849:Chromatography, Gas; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D005942:Gluconates / Q000737:chemistry / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008855:Microscopy, Electron, Scanning; D009249:NADP / Q000378:metabolism; D010755:Organophosphates / Q000737:chemistry; D010421:Pentanoic Acids / Q000737:chemistry; D010766:Phosphorylation; D011091:Polyesters / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature; D016963:Thermus thermophilus / Q000378:metabolism*; D013997:Time Factors
D002210:Caprylates; D002264:Carboxylic Acids; D005942:Gluconates; D010755:Organophosphates; D010421:Pentanoic Acids; D011091:Polyesters; C013056:beta-hydroxyvaleric acid; D009249:NADP; C011632:acetyl phosphate; D002244:Carbon; C101940:3-hydroxyoctanoic acid; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; D000102:Acetyl-CoA C-Acyltransferase; D000469:Alkaline Phosphatase; D005033:Ethylmaleimide; C031492:octanoic acid; C030691:gluconic acid; D020155:3-Hydroxybutyric Acid