Functional andStructural Analyses ofThreonine Dehydratase fromCorynebacterium glutamicum (1992)

Threonine dehydratase activity isanimportant element intheflux control ofisoleucine biosynthesis. The enzyme ofCorynebacterium gluamicum demonstrates amarkedsigmoidal dependence ofinitial velocity onthe threonine concentration, adependence thatisconsistent withsubstrate-promoted conversion oftheenzyme fromalow-activity toahigh-activity conformation. Inthepresence ofthenegative aliosteric effector isoleucine, theK.45 increased from21to78mM andthecooperativity, asexpressed bytheHill coefficient increased from 2.4to3.7. Valine promoted opposite effects: theK0.5 wasreduced to12mM,andtheenzyme exhibited almost nocooperativity. Sequence determination oftheC.ghutamicum geneforthis enzyme revealed anopenreading framecoding fora polypeptide of436aminoacids. Fromthisinformation andthemolecular weight determination ofthenative enzyme, itfollows that thedehydratase isatetramer withatotal massof186,396 daltons. Comparison ofthededuced polypeptide sequence withthesequences ofknownthreonine dehydratases revealed surprising differences fromtheC.glutamicum enzymeinthecarboxy-terminal portion. Thisportion isgreatly reduced insize, andalarge gapof95aminoacids mustbeintroduced toachieve homology. Therefore, theC.glutamicum enzymemustbeconsidered asmall variant ofthreonine dehydratase thatis typically controlled byisoleucine andvaline buthasanaltered structure reflecting atopological difference in theportion oftheprotein mostlikely tobeimportant foraliosteric regulation. Threonine dehydratase (EC4.2.1.16) catalyzes theconversion ofthreonine to2-oxobutyrate, ammonium,and water. Theanabolic enzymeinitiates asequence offive reactions leading totheultimate formation ofisoleucine. Furthermore, itistheonlyenzymespecific forisoleucine synthesis. Therefore, thedehydratase isanideal target for thestudyofthefluxcontrol ofisoleucine biosynthesis. Indeed, theenzymeofEscherichia coli wasthefirst example ofanenzymeatthebeginning ofabiosynthetic sequence thatissubject toend-product inhibition (47). Workonthis enzymebyUmbarger etal.(21, 48)andChangeux (6)has decisively influenced theallosteric regulation modelof Monodetal.(33). Inallthreonine dehydratases studied sofar, isoleucine is thenegative allosteric effector andvaline isthepositive allosteric effector. Isoleucine increases cooperativity, which ismanifested asa sigmoidal dependence ofactivity in substrate saturation kinetics. Several aspects, however, are notwellunderstood. Thus,without anyeffector forthe enzymeofE.coli, sigmoidal dependence hasbeendescribed (14, 48), whereas fortheenzymes ofSalmonella typhimurium(12), Bacillus subtilis (20), andyeast cells (4), hyperbolic kinetics havebeennoted. Threonine dehydratases are tetramers ofidentical subunits (8,27), butitisnotknown howligand binding promotes structural changes toeffect substrate binding. Possibly, thenumberofbinding sites, pyridoxal 5'-phosphate content, andtheoligomeric state are involved, asshownfortheenzymeofS.typhimurium (19, 23). Thereareseveral indications frommutant enzymes ofE. coli andahomology comparison ofthebiosynthetic threoninedehydratase (ilvA) withitscatabolic counterpart (tdc) thatthecarboxy-terminal portion isinvolved inallosteric regulation oftheenzyme(10, 15,45). Sincebiosynthetic threonine dehydratase activity is