Biomedical Engineering Reference
In-Depth Information
acids, which are incorporated into the polypeptide chains of nascent proteins. The
rate of admission of acetyl-CoA into TCA cycle is dependent on the availability of
sources of nitrogen, phosphorous, and other elements, as well as on the oxidative
potential of the environment [6].
In Figure 2.2, Pathway II, limitations in nitrogen, phosphorous, oxygen [77, 78],
magnesium, or sulfate [79] lead to P(3HB) production. This limitation causes ces-
sation of protein synthesis leading to high concentrations of NADH and NADPH
resulting in an inhibition of citrate synthase and isocitrate dehydrogenase and in
a slowdown of the TCA cycle and the channeling of acetyl-CoA toward P(3HB)
biosynthesis [35]. Acetyl-CoA no longer enters the TCA cycle at the same rate and
instead is converted to acetoacetyl-CoA by 3-ketothiolase, the fi rst enzyme of the
P(3HB) biosynthetic pathway, which is inhibited by CoA.
According to Figure 2.2, Pathway II, three enzymes are involved in PHA
SCL
production: 3-ketothioloase; acetoacyl-CoA reductase, and PHA synthase. The role
of these enzymes is described below.
The fi rst step for PHA formation is catalyzed by
3 - ketothiolase
. Its mechanism
includes two partial reactions which result in a condensation of two acetyl-CoA
molecules to obtain acetoacyl-CoA. Two cystein residues are present in the active
site of this enzyme and are responsible for the acetyl-CoA molecule ligament at
the enzyme and for the activation of a second molecule of acetyl-CoA, hence entail-
ing a condensation and formation of acetoacyl-CoA [1]. The enzyme catalyzes the
reversible reaction shown in Eq. (2.1):
2 Acetyl-CoA
↔
acetoacyl-CoA
+
CoASH
(2.1)
This 3-ketothiolase competes for acetyl-CoA with many other metabolic pathways,
including acetate, citrate, and the fatty acids synthesis. This enzyme is inhibited
by free CoASH molecules [80].
Acetoacyl - CoA reductase
catalyzes the second step on PHA biosynthesis (Eq.
(2.2) ), converting acetoacyl - CoA into hydroxyacyl [1] :
Acetoacyl-CoA
+
NADPH
+
H
+
↔
3-hydroxyacyl-CoA
+
NADP
+
(2.2)
Two acetoacyl-CoA reductase types, with different specifi cities for substrates and
coenzymes, were found in
C. necator
. The NADH-dependent enzyme is active in
D(
) substrates, while a NADPH-dependent one is stereospecifi c or active
only at C4 to C6 D(
−
) and L(
+
)3 - hydroxyacyl - CoA substrates. During the P(3HB) synthesis,
acetoacetyl-CoA is reduced to D(
−
−
)3 - hydroxybutyryl - CoA, catalyzed by NADPH -
dependent enzymes [5] .
PHA synthase
is the key enzyme for PHA biosynthesis. This enzyme catalyzes
ester formation through the polymerization of D(
)3 - hydroxyacyl - CoAs units,
resulting in the polymer. The wide monomer variety that composes PHA is related
to large substrate PHA synthase specifi cities. In this context,
C. necator
PHA
synthase is able to polymerize 3-hydroxy, 4-hydroxy, and 5-hydroxyalkanoates from
the 4 and 5 carbon hydroxyacyl-CoA, D-isomers [5, 12]. This enzyme is shown in
−
