Inhibition Ketothiolase

If nitrogen (in the form of ammonia) is growth limiting, the potential applications of acetyl-CoA and NAD(P)H are restricted. Liberated NAD(P)H cannot be consumed for reductive syntheses, for instance of amino acids, it remains available and starts to inhibit citrate synthase [45, 46]. To the extent that the TCA cycle is thereby inhibited, acetyl-CoA should become available for the 3-ketothiolase, and could flow into poly(3HB) (Fig. 1, Table 1). 

The rate of fatty acid oxidation is most likely regulated by the carnitine-dependent transport of acyl residues across the mitochondrial inner membrane, the supply of fatty acids to the cell, and the concentration of cofactors such as CoA and carnitine. Moreover, feedback inhibition is exerted by NADH on 3-hydroxyacyl-CoA dehydrogenase and by acetyl-CoA on 3-ketothiolase. Interestingly, malonyl-CoA, an intermediate in the fatty acid biosynthetic pathway, is a strong inhibitor ofCAT-I. 

Free CoA is inhibiting 3-ketothiolase as a consequence, the carbon flux towards PHAs is decreased. To a lower extent, 3-ketothiolase is also inhibited by NAD+ and ATP. Additionally, high amounts of NAD + slow down the reduction steps from acetoacetyl-CoA to 3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase. 

Poly([f ]-3-hydroxybutyrate) degradation is regulated by inhibition of the oxidative conversion of 3-hydroxybutyrate to acetoacetate. Here, NADH2, pyruvate and oxalacetate act as inhibitors. The last step, the splitting of acetoacetyl-CoA into two acetyl-CoA molecules by 3-ketothiolase, is product inhibited by high concentrations of acetyl-CoA. High concentrations of free CoA, the second substrate of this cleaving reaction, relieve this product inhibition [28]. 

Bromooctanoate, by becoming converted to 2-bromo-3-ketooctanoyl-CoA, irreversibly inhibits the 3-ketothiolase (EC 2.3.1.6) activity (Raaka and Lowenstein, 1979). 4-Bromooctanoate does likewise by becoming converted to 4-bromo-3-ketobutyryl-CoA (Olowe, 1981). Arsenite restrains p-oxi-dation by inhibiting acetoacetyl-CoA thiolase (EC 2.3.1.9) (Rein et a/., 1979). The inhibitory effect of pent-4-enoate on the activities of 3-ketoacyl-... 

Intracellular degradation occurs via hydrolytic cleavage [7] and 3-ketothiolase is the enzyme that plays a key role in the production and degradation of PHA. The presence of a limited carbon source in the medium stimulates the process of degradation. In these conditions, the level of acetyl-coenzyme A (CoA) increases and prevents the inhibition of 3-ketothiolase by acetoacetyl-CoA. This allows the release of acetyl-CoA from P(3HB) and inhibits the condensation of acetyl-CoA to acetoacetyl-CoA [8]. PhaZ enzymes are secreted in order to break down the polymer into hydroxyacids which are then ntilised by the microorganism as a carbon source for growth [9]. Once... 

PHB synthesis from glucose using Azotobacter beijerinkii revealed substantial amounts of polymer accumulation under oxygen limitation conditions. The key feature of control in A. beijerinckii is the pool size of acetyl-CoA, which may either be oxidized via the tricarboxylic acid (TCA) cycle or can serve as a substrate for PHB synthesis the diversion depends on environmental conditions, especially oxygen limitation, when the NADH/NAD ratio increases. Citrate synthase and isocitrate dehydrogenase are inhibited by NADH, and as a consequence, acetyl-CoA no longer enters the TCA cycle at the same rate. Instead acetyl-CoA is converted to acetoacetyl-CoA by p-ketothiolase, the first... 

PHB is produced from acetyl-CoA by the sequential action of three enzymes, 3-ketothiolase, acetoacetyl-CoA reductase and PHA synthase (Pathway I) [29]. 3-ketothiolase reversibly combines two acetyl-CoAs into acetoacetyl-CoA and is competitively inhibited by high concentrations of CoASH, which is released when acetyl-CoA enters the TCA cycle [140]. NADPH-dependent acetoacetyl-CoA reductase... 

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