Page 74 - Biodegradable Polyesters

52 3 Microbial Synthesis of Biodegradable Polyesters: Processes, Products, Applications
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of synthases. This enzyme could be differentiated by heat stability up to 60 C,
wherein, the enzyme still exhibited about 90% of the maximum enzyme activity,
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attained at 40 C. The soluble archaeal PHB synthase was only active at high salt
concentration, while the granule-bound PHB synthase was almost independent of
the salt concentration.
There are no current structural data of polyester synthases. The secondary
structure content was inferred by predictions implementing the multiple align-
ments of synthases. The use of JPred indicated that polyester synthases are mostly
composed of variable loops (49.7%) and α-helical (39.9%) secondary structures,
whereas only 10.4% were proposed as β-sheet secondary structures [38]. How-
ever, circular dichroism spectroscopy suggested that the class II synthase from P.
aeruginosa is comprised of the secondary structures: 10% α-helix, 50% β-sheet,
and 40% random coil [39].

3.5
Catalytic Reaction Mechanism

In all of the structural models, the amino acid residues apparently constituting the
catalytic triad or involved in covalent catalysis were identified as being adjacent
to the core structure with the putative active site nucleophile cysteine located at
the elbow of the strand-elbow helix motif. In the class II polyester synthase, the
highly conserved histidine residue which functions as a general base catalyst in
α/β-hydrolases was functionally replaced by an adjacent histidine residue, which
too was close to the core structure.
PHA synthases have a cysteine residue as catalytic nucleophile and because
of this, the general base catalyst histidine would be sufficient for nucleophilic
activation as has been shown for cysteine proteases [40]. The aspartate-302-
alanine mutant of the A. vinosum class III synthase has now been studied in
greater detail. The in vitro results would again suggest a more important purpose
of aspartate-302 in elongation than activation of the catalytic cysteine. Tian
et al. [41] also demonstrated by using differing substrate to enzyme ratios and
monitoring PHB formation at PhaC that the synthase initiates polymerization
through self-priming. It was proposed that the synthase has the capacity of chain
termination and re-initiation. In polyester synthases, the second general base
catalyst (aspartate) is required to activate the 3-hydroxyl of the 3-hydroxybutyryl-
CoA or the bound 3-hydroxybutyryl to enable nucleophilic attack on the acylated
enzyme and/or self-priming (Figure 3.4).
What was the initially postulated catalytic mechanism based on the catalytic
reaction mechanism of fatty acid synthases [42] has been advanced, considering
a reaction mechanism found in α/β-hydrolases. Two thiol groups now provided
by cysteine residues are considered being a part of covalent catalysis. One thiol
group serves as the accepting site for the substrate 3-hydroxybutyryl-CoA, while
the second thiol group serves for priming and elongation. There is some evidence
supporting the fatty acid synthase mechanism such as (i) the requirement of the