download report - Istituto Pasteur

P a r t i c i p a n t s :
Alessandra Bonamore, Alberto Macone, post-doc fellows.
C o l l a b o r a t i o n s :
Dipartimento di Chimica, Università di Firenze (Prof. Giulietta
Smulevich), CPC Biotech srl, Napoli (Dr. Fabio Arenghi).
Report of activity
The main target of the project is to attribute biochemical
and physiological functions to the vast family
of bacterial globins and exploit their versatile
catalytic properties in biotransformation processes
for the synthesis of valuable intermediates for biopharmaceutical
applications. To this end, the
research project entails the identification of novel
bacterial hemoglobins and their cloning into engineered
Escherichia coli strains bearing oxygen
dependent enzymes of biotechnological interest. In
the first part of the project (see previous report of
activity), the alkylhydroperoxide reductase of flavohemoglobins
has been exploited for the modification
of phospholipid acyl chains in order to produce novel
substituted fatty acids. Moreover, this enzymatic
activity of bacterial globins, flavohemoglobins in
particular, has been demonstrated to be a major
determinant in the physiological response to oxidative
stress in bacteria. This finding has been recognized
as a key biochemical clue for the understanding
the physiological role of flavohemoglobins. In the
last year of the project, the research has been focused
mainly on truncated hemoglobins and in particular
to their functional role.
Novel bacterial globins
Novel chimeric proteins made of a globin domain
fused with a “cofactor free” monooxygenase domain
have been identified within the Streptomyces avermitilis
and Frankia sp. genomes by means of bioinfor-
Principal investigator: Alberto Boffi
Professor of Molecular Biology
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”
Tel. +39 06 49910990; Fax +39 06 4440062
alberto.boffi@uniroma1.it
5
Molecular biology of microorganisms and viruses - AREA 1
Escherichia coli strains overexpressing flavohemoglobins for the
production of novel, biologically active compounds derived from
phospholipid post-biosynthetic modifications
matics methods. Structure based sequence alignments
show that the globin domains of both proteins
can be unambiguously assigned to the truncated
hemoglobin family, in view of the striking similarity
to the truncated hemoglobins from Mycobacterium
tuberculosis, Thermobifida fusca and Bacillus subtilis. In
turn, the non-heme domains belong to a family of
small (about 100 aminoacids) homodimeric proteins
annotated as antibiotic biosynthesis monooxygenases,
despite the lack of a cofactor (e.g., a metal, a
flavin or a heme) necessary for oxygen activation.
The chimeric protein from S. avermitilis has been
cloned, expressed and characterized. The protein is a
stable dimer in solution based on analytical ultracentrifugation
experiments. The heme ligand binding
properties with oxygen and carbonmonoxide resemble
those of other truncated hemoglobins. In addition,
an oxygen dependent redox activity has been
demonstrated towards easily oxidizable substrates
such as menadiol and p-aminophenol. These findings
suggest novel functional roles of truncated hemoglobins,
which might represent a vast class of multipurpose
oxygen activating/scavenging proteins
whose catalytic action is mediated by the interaction
with cofactor free monooxygenases.
Understanding active site dynamics in
truncated hemoglobins
The heme binding pocket of truncated hemoglobins
is shaped by a triad of aminoacids, tipically a tyrosine
(TyrB10), a tryptophane (TrpG8) and a phenylalanine
(PheCD1). The specific role of these
aminoacids in heme ligand binding and catalysis has
not been unveiled as yet. Thus, the active site of the
oxygen-avid truncated hemoglobin from Bacillus subtilis
has been characterized by infrared absorption
and resonance Raman spectroscopies, and the
dynamics of CO rebinding after photolysis has been
investigated by picosecond transient absorption
spectroscopies. The very low C-O stretching fre-