 The
overall objective of this
project is to provide detailed
information about the structure
and function of a group of
aminoglycoside modifying
enzymes, the aminoglycoside
6-phosphotransferases,that
confer resistance to streptomycin
or its close relatives. Streptomycin
acts by binding to the A
site of the 16S ribosomal
RNA molecule; this blocks
translocation. The
6-phosphotransferases inactivate
the antibiotic by catalyzing
the ATP-dependent phosphorylation
of streptomycin at position
6 of the streptidine ring.
The
focus is on two members of
this group: the enzyme APH
(6)-1a, whose gene is found
within the streptomycin biosynthetic
cluster of Streptomyces griseus,
a common soil bacterium;
and the enzyme APH(6)-ld,
found on the broad host-range
plasmid RSF1010 within human
pathogenic bacterial isolates.
The rationale for selecting
these particular enzymes,
APH(6)-la and –ld,
is that, although similar
in terms of the product they
generate, they are different
in terms of their source,
i.e., non-pathogenic versus
pathogenic bacteria, and
their ecological role. It
is expected that a comparison
between them and with other
aminoglycoside phosphotransferases
will yield important information
about what, if anything,
mechanistically and structurally
distinguishes the enzyme
from nonpathogenic (S. griseus)
and pathogenic (E. coli and
others) bacteria.
The
specific aims are to:
- obtain
the genes for the two
enzymes, express their
gene products in Escherichia
coli, and characterize
them in terms of their
regiospecificity using
NMR and their detailed
kinetic mechanisms applying
steady-state kinetics;
- identify
the key amino acid residues
of the two enzymes that
are involved in catalysis,
ATP binding, streptomycin
binding, and domain association;
and
- determine
the three dimensional
structure of either APH(6)-Ia
or -Id.
The
detailed information gathered
here about the structure
and function of the streptomycin
6-phosphotransferases will
extend our overall understanding
of how aminoglycoside phosphotransferases
bind and modify their substrates,
which will aid in the design
of general inhibitors of
this class of resistance
enzymes. Insights gained
in this work also may give
clues about how aminoglycoside
resistance spreads within
bacterial communities. This
project utilizes resources
of both the BNMR laboratory
and the LMCB.
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