Physics And Chemistry Basis Of Biotechnology - De Cuyper - tiera.ru

Rational design of P450 enzymes for biotechnology
In the absence of P450 2E1 crystal structure its active site conformation is not known.
However, studies on its different substrates and competitive inhibitors, have provided
useful information on some of the properties of its active site including:
it binds and efficiently oxidises small molecules,
accommodates water-soluble molecules although hydrophobicity is an
important feature of the substrate-binding pocket,
does not effectively accommodate molecules with a formal ionic charge [ 146].
This section reports on the modelling, construction and expression of a chimeric protein
designed to confer the human P450 2E1 new and improved properties, like solubility
and self-sufficiency in catalysis, hence conferring the ability of receiving electrons
directly from small electron donors (NADPH), maintaining, at the same time, its quite
peculiar substrate specificity. Self-sufficient and soluble cytochrome P450 systems are,
in fact, essential to employ the catalytic power of these enzymes for biotechnological
purposes.
The chimeric protein was obtained by fusing part of the human P450 2E1 with a
portion of P450 BM3. This latter cytochrome exhibits key features, as mentioned
earlier, that make it an ideal candidate for fusion with the human P450 2E1:
it is catalytically self-sufficient due to the presence of a reductase domain
within the same polypeptide chain, containing both FMN and FAD and
requiring only NADPH for activity,
it is highly homologous to the human P450 enzymes (30% homology between
the P450 BM3 haem domain and 2E1), and it shares many common features
leading to the classification in the same class II as the human microsomal P450
enzymes.
Furthermore, the three-dimensional structure of P450 BM3 has been solved [46, 147],
its gene has been cloned [66] and its catalytic properties have been studied.
4.1. MODELLING
Previous available models of mammalian P450s were often approximate since they
were based either solely on the structure of P450cam, that has a very low sequence
identity with mammalian enzymes (from ca 15 to 20%) [148], or on models of the
binding site which is expected to be the most variable region of the protein. Only
recently, improved models were obtained after observing the necessity to generate a
multiple sequence alignment of the target P450 with template proteins. These
alignments should be done by looking at the structurally conserved regions of the
templates and not simply relying on automated alignment procedures, since these are
often not reliable, especially for some regions of the protein [148].
To help the design of a new valid chimera likely to possess the desired properties, a
three-dimensional model of P450 2E1 was built in this laboratory. The P450 2E1 model
was generated using a Silicon Graphics Indigo2 IRIX 6.2 workstation equipped with
the Biosym/MSI software Insight. The following protocol was used for the designing of
the model:
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