Download Mutants, metals and mechanism of 5

Biochemical Society Transactions (2002) Volume 30, Part 3
A47
M1 Antibodies: a paradigm for the evolution of molecular
A2 Mutants, metals and mechanism of 5-aminolaevulinic acid
recognition
Michael NeuberPer
Medical Research Council Laboratory of Molecular Biology, Hills
Road, Cambridge CB2 2QH, UK.
deh ydratase
P.M.Shoolinein-lordan
Biochemistry and Molecular Biology, School of Biological Sciences,
Southampton University, Bassett Crescent East, Southampton, SO16
7PX, UK
-
The generation of novel proteins and the modification of old ones
lies at the very heart of biological evolution. Genes encoding novel
proteins are produced by gene duplication, gene conversion and
point mutations; those mutant proteins conferring an advantage are
selected. The process takes a long time.
The humoral immune system also functions by the production and
selection of novel proteins. During the course of an immune
response, new antibodies are generated (which never existed before)
and those recognising a foreign antigen with high affinity are
selected. As in germline evolution of proteins, the somatic
evolution of antibodies also relies on gene diversification through
mutation followed by selection. However, in the case of somatic
antibody evolution, the mutational process is localised (targeted to
the antibody genes themselves) and the novel antibodies are
generated in a period of days rather than millions of years. In this
lecture I will review the mutational processes (localised hypermutation and gene conversion) that underpin the generation of high
affinity antibodies and will discuss the mechanisms by which
improved antibodies are selected in vivo. I will then illustrate how
our studies into the natural mechanism of antibody production in
vivo allows us to design systems in which we can recapitulate the
evolution of high affinity binding sites in vitro.
The enzymic synthesis of porphobilinogen, the pyrrole precursor of
all tetrapyrroles, is catalysed by 5-aminolaevulinic acid dehydratase
(ALAD) in a reaction involving the dimerisation of two molecules
of 5-aminolaevulinic acid (ALA). The active sites of all ALADs
possess individual binding sites for each of the two ALA molecules,
termed the A-site and the P-site. Despite the similarities between
the amino acid sequences of ALADs from bacteria, plants and
animals, ALADs fall into two broad classes - those that use Zn2+
ions and those that use Mg2+ ions. Metal substitution experiments
and mutations at the metal binding site, supported by X-ray crystallography, have provided insight into the role of the metal in the
catalytic mechanism and in the nature of the powerful inhibition of
the Zn2+ enzymes by Pb2+. Mutations of active site amino acid
residues, notably two catalytic lysines, have also established the
identity of functional groups important for substrate binding and
porphobilinogen formation. X-Ray structures of ALAD with
bound substrate/s, substrate analogues, inhibitors and the product
porphobilinogen have provided detailed insight into the detailed
architecture of the A- and P-substrate binding sites, the catalytic
mechanism and the molecular pathology of human ALAD
deficiency diseases.
A1 Structure and function of glutamyl-tRNA reductase involved
A3 The terminal steps of heme biosynthesis
in 5-aminolevulinic acid formation
J. Moserl, W.-D. Schubert2, D. Heinz2 and D. J a h n l
1 Institute for Microbiology, Technical University
Braunschweig,Spielmannstr.7 , D 38106 Braunschweig, Germany 2
Department of Structural Biology, German Research Center of
Biotechnology, Mascheroder Weg 1, D 38104 Braunschweig,
Germany
H.A. Dailey
Biomedical and Health Sciences Institute, A222 Life Sciences
Building, University of Georgia, Athens, GA 30602-7229 USA
In most bacteria, in archaea and in plants the general precursor of all
tetrapyrroles, 5-aminolevulinic acid, is formed by two enzymes. The
initial substrate glutamyl-tRNA is reduced by NADPH-dependent
glutamyl-tRNA reductase to form glutamate-1 -semialdehyde. The
aldehyde gets subsequently transaminated by glutamate-l-semialdehyde-2,1 -aminomutase to yield 5-aminolevulinic acid. The
enzymatic mechanism and the solved crystal structure of
Methanopyrrus kandleri glutamyl-tRNA reductase will be
described. A pathway for metabolic channeling of the reactive
aldehyde between glutamyl-tRNA reductase and the aminomutase
will be proposed.
Moser et al. (1999) J. Biol. Chem. 274, 30679-30685.
Moser et al. (2001) EMBO J. 20,6583-6590.
The terminal three steps in heme biosynthesis are the oxidative
decarboxylation of coproporphyrinogen I11 to protoporphyrinogen
IX, followed by the six electron oxidation of the protoporphyrinogen to protoporphyrin IX and finally the insertion of iron to form
protoheme IX. Interestingly, nature has evolved distinct enzymatic
machinery to deal with the antepenultimate (coproporphyrinogen
oxidase) (CP0)and penultimate (protoporphyrinogen oxidase)
(PPO) steps for aerobic vs. anaerobic organisms. The terminal step
is catalyzed by an enzyme, ferrochelatase. This enzyme is clearly
conserved with regard to a small set of essential catalytic residues,
but varies significantly with regard to size, subunit composition,
cellular location, and the presence o r absence of a [2Fe-2S] cluster.
C P O and PPO will be reviewed with regards to their enzymatic and
physical characteristics. Ferrochelatase, the best characterized of
these three enzymes, will be characterized with particular emphasis
paid to what has been learned from the crystal structure of the B.
subtilis and human enzymes.
0 2002 Biochemical Society