Cob
al
amin-dependent methionine synth
ase from
Escherichia coli is
a monomeric 136 kD
aprotein composed of multiple function
al regions. The X-r
aystructure of the cob
al
amin-binding region ofmethionine synth
ase reve
als th
at the cof
actor is s
andwiched between
an
![](/im<font color=)
ages/gifch
ars/
alph
a.gif" BORDER=0>-helic
al dom
ain th
at cont
actsthe upper f
ace of the cob
al
amin
and
an
![](/im<font color=)
ages/gifch
ars/
alph
a.gif" BORDER=0>/
![](/im<font color=)
ages/gifch
ars/bet
a2.gif" BORDER=0 ALIGN="middle"> (Rossm
ann) dom
ain th
atinter
acts with the lower f
ace. Anunexpected conform
ation
al ch
ange
accomp
anies binding of themethylcob
al
amin cof
actor. The dimethylbenzimid
azole lig
and to the lower
axi
al position of the cob
alt in thefree cof
actor is displ
aced by histidine759 from the Rossm
ann dom
ain [Drenn
an, C. L., Hu
ang, S., Drummond, J.T., M
atthews, R. G., & Ludwig,M. L. (1994)
Science 266, 1669]. In orderto f
acilit
ate studies of the roles of
amino
acid residues inthecob
al
amin-binding region of methionine synth
ase, we h
ave constructed
asynthetic module correspondingto nucleotides (nt) 1741-2668 in the
metH gene
andincorpor
ated it into the wild-type
metH gene.Thismodule cont
ains unique restriction sites
at ~80 b
ase p
air interv
als
and w
as synthesized by overl
ap extensionof 22 synthetic oligonucleotides r
anging in length from 70 to 105 nt
and subsequent
amplific
ation usingtwo sets of primers. Expression of methionine synth
ase from
apl
asmid cont
aining the modified genew
as shown to be un
affected by the introduction of the synthetic module.
E. coli does not synthesizecob
al
amin,
and overexpression of MetH holoenzyme requires
acceler
atedcob
al
amin tr
ansport. Growthconditions
are described th
at en
able the production of holoenzymer
ather th
an
apoenzyme. We describethe construction
and initi
al ch
ar
acteriz
ation of seven mut
ants.Four mut
ations (His759Gly, Asp757Glu,Asp757Asn,
and Ser810Al
a)
alter residues in the hydrogen-bonded networkHis-Asp-Ser th
at connectsthe histidine lig
and of the cob
alt to solvent. Three mut
ations(Phe708Al
a, Phe714Al
a,
and Leu715Al
a)
alter residues in the c
ap region th
at covers the upper f
ace of thecob
al
amin. The His759Gly mut
ation h
asprofound effects, essenti
ally
abolishing ste
ady-st
ate
activity, whilethe Asp757, Ser810, Phe708,
and Leu715mut
ations le
ad to decre
ases in
activity. These mut
ations
assessthe import
ance of individu
al residues inmodul
ating cob
al
amin re
activity.