Structures of the N-terminal modules imply large domain motions during catalysis by methionine synthase

105

109

Lysine 5,6-aminomutase is an adenosylcobalamin and pyridoxal-5 -phosphate-dependent enzyme that catalyzes a 1,2 rearrangement of the terminal amino group of DL-lysine and of L-␤-lysine. We have solved the x-ray structure of a substrate-free form of lysine-5,6-aminomutase from Clostridium sticklandii. In this structure, a Rossmann domain covalently binds pyridoxal-5 -phosphate by means of lysine 144 and positions it into the putative active site of a neighboring triosephosphate isomerase barrel domain, while simultaneously positioning the other cofactor, adenosylcobalamin, Ϸ25 Å from the active site. In this mode of pyridoxal-5 -phosphate binding, the cofactor acts as an anchor, tethering the separate polypeptide chain of the Rossmann domain to the triosephosphate isomerase barrel domain. Upon substrate binding and transaldimination of the lysine-144 linkage, the Rossmann domain would be free to rotate and bring adenosylcobalamin, pyridoxal-5 -phosphate, and substrate into proximity. Thus, the structure embodies a locking mechanism to keep the adenosylcobalamin out of the active site and prevent radical generation in the absence of substrate.A denosylcobalamin (AdoCbl; coenzyme B 12 ) is nature's biochemical radical reservoir, capable of catalyzing challenging chemical reactions by way of H atom abstraction and the generation of free-radical intermediates (1-3). AdoCbldependent isomerases catalyze 1,2 shifts between an H atom and a functional group such as -OH, -NH 3 ϩ , -(CO)S-coenzyme A, or other carbon-based groups. The catalytic power of AdoCbl lies in the homolytic cleavage of its weak (Ϸ30 kcal͞mol) organometallic C-Co bond, formed between an octahedral Co(III) center with five N coordinations and a 5Ј-deoxyadenosyl (Ado) axial ligand. C-Co bond homolysis results in the transient formation of cob(II)alamin and 5Ј-deoxyadenosyl radical (Ado • ). Ado • abstracts an H atom from the substrate, forming a substrate radical and 5Ј-deoxyadenosine (AdoH). To close the catalytic cycle, substrate reabstracts the H atom from AdoH, and recombination of cob(II)alamin and Ado • accompanies product formation. Amazingly, the enzymatic rate of C-Co bond homolysis is enhanced by a factor of Ϸ10 12 over nonenzymatic homolysis (4, 5). AdoCbl-dependent isomerases are often present in catabolic pathways and can serve to rearrange the substrate's carbon skeleton and͞or functional groups for further degradation. One such pathway that operates in several bacterial species is the fermentation of lysine to yield acetate. Interestingly, the lysine fermentation pathway contains two analogous enzymes: lysine 5,6-aminomutase (5,6-LAM), which is AdoCbl-dependent (6, 7), and lysine 2,3-aminomutase (2,3-LAM), which is an S-adenosylmethionine (AdoMet or SAM)-dependent ironsulfur enzyme (8-10). Both enzymes require pyridoxal 5Ј-phosphate (PLP) (8, 11) in addition to AdoCbl or AdoMet, and both catalyze a 1,2 amino group shift with concomitant H atom migration (Fig. 1A). In 5,6-LAM, AdoCbl is the source of the transient Ado • , whereas, in 2,3-L...

Section: Resultsmentioning

confidence: 58%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

Berkovitch

Behshad

Tang

et al. 2004

105

109

“…Because of the high abundance of (␤␣) 8 -barrelcontaining proteins, many structures were selected. The highest hit with both subunits of CoFeSP Ch (DALI Z-score of 23) was observed with residues 354-649 of cobalamin-dependent methionine synthase (18). For a superimposition of 230 C␣-atoms of CfsB and methionine synthase, an rmsd value of 2.5 Å was obtained.…”

Section: Resultsmentioning

confidence: 99%

Structural insights into methyltransfer reactions of a corrinoid iron–sulfur protein involved in acetyl-CoA synthesis

Svetlitchnaia¹,

Svetlitchnyi²,

Meyer

et al. 2006

106

146

acety-CoA pathway ͉ Carboxydothermus hydrogenoformans ͉ methyltransferase ͉ cobalt B 12-dependent enzymes are widespread in nature and catalyze distinct reactions. They can be grouped in three major classes: the isomerases, reductive dehalogenases, and methyltransferases (MeTrs; ref. 1). The cobalamin-dependent MeTrs play important roles in the amino acid biosynthesis in many organisms, ranging from bacteria to humans, as well as in the one-carbon metabolism of bacteria and archaea. MeTrs typically catalyze the transfer of methyl groups between organic and inorganic donors and acceptors like S-adenosylmethionine, methyltetrahydrofolate, and cobalamins. A unique methyltransfer reaction is found within the acetyl-CoA (Ljungdahl-Wood) pathway of autotrophic carbon assimilation, which operates in the anaerobic acetogenic, sulfidogenic, methanogenic, and hydrogenogenic bacteria and archaea. In the final step of the pathway, acetyl-CoA is synthesized from a methyl cation, carbon monoxide (CO), and CoA, a reaction catalyzed by the Ni-and Fe-containing enzyme acetyl-CoA synthase (ACS; for a review, see ref.2). Key to this reaction (Eq. 1) is the action of the Coand Fe-containing corrinoid iron-sulfur protein (CoFeSP) that donates the methyl group from methyl-cob(III)amide to one of the two Ni ions in the active site cluster A of ACS (3). This is the only methyltransfer reaction known with metals as donors and acceptors of the methyl group: CH 3 OCoFeSP ϩ CO ϩ CoA 3 CH 3 (CO)CoA ϩ CoFeSP. [1]CoFeSP has been isolated and characterized from the acetogenic bacterium Moorella thermoacetica (4), the methanogenic archaeon Methanosarcina thermophila (5), and the hydrogenogenic bacterium Carboxydothermus hydrogenoformans (6).C. hydrogenoformans can use CO as a sole source of energy and carbon under anaerobic chemolithoautotrophic conditions. Protons serve as terminal electron acceptor (7), and the methyl branch of the acetyl-CoA pathway is used for the assimilation of carbon (6). The oxidation of CO in this bacterium is catalyzed by two monofunctional NiFe-containing CODHs (CODHI Ch and CODHII Ch ) that harbor the [Ni-4Fe-5S] active site cluster C (7). A monomeric ACS Ch , a 1:1 molar complex of ACS Ch and CODHIII Ch , and CoFeSP Ch are expressed in functional form during the growth of C. hydrogenoformans using CO as the only substrate (6). The CoFeSP Ch from C. hydrogenoformans is a heterodimeric protein composed of a large subunit (48.4 kDa, CfsA) and a small subunit (33.9 kDa, CfsB) and harbors one molecule of the cobalt-containing corrinoid cofactor and a single The CoFeSPs from acetogens and methanogens are heterodimeric proteins sharing sequence identities of 35-53% with CoFeSP Ch from C. hydrogenoformans. CoFeSP Mt from M. thermoacetica was shown to contain two cofactors, 5-methoxybenzimidazolylcobamide (a variant of B12) and a [4Fe-4S] 2ϩ/1ϩ cluster, which gave the protein its name. For the cobamide cofactor, it was shown that neither the 5-methoxybenzimidazolyl group nor a nitrogen atom from a protein side chain is coor...

“…Thus, substantial domain reorganization is necessary for the ACP domain and its covalently tethered substrates to interact successively with first the AT and then the KS domain of the paired subunit of the DEBS module. Similar requirements for major conformational changes have been predicted in other multienzyme systems (30,31). How the requisite quaternary structural changes that accompany catalysis are achieved remains unknown at present and is the subject of ongoing investigations into the structure and mechanism of modular PKS megasynthases.…”

Section: Interaction Between the Acp Domain And The Ks-at Fragment: Notmentioning

confidence: 92%

The 2.7-Å crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B synthase

Tang

Kim

Mathews

et al. 2006

258

412

The x-ray crystal structure of a 194-kDa fragment from module 5 of the 6-deoxyerythronolide B synthase has been solved at 2.7 Å resolution. Each subunit of the homodimeric protein contains a full-length ketosynthase (KS) and acyl transferase (AT) domain as well as three flanking ''linkers.'' The linkers are structurally well defined and contribute extensively to intersubunit or interdomain interactions, frequently by means of multiple highly conserved residues. The crystal structure also reveals that the active site residue Cys-199 of the KS domain is separated from the active site residue Ser-642 of the AT domain by Ϸ80 Å. This distance is too large to be covered simply by alternative positioning of a statically anchored, fully extended phosphopantetheine arm of the acyl carrier protein domain from module 5. Thus, substantial domain reorganization appears necessary for the acyl carrier protein to interact successively with both the AT and the KS domains of this prototypical polyketide synthase module. The 2.7-Å KS-AT structure is fully consistent with a recently reported lower resolution, 4.5-Å model of fatty acid synthase stucture, and emphasizes the close biochemical and structural similarity between polyketide synthase and fatty acid synthase enzymology. modular megasynthase ͉ multienzyme assembly ͉ polyketide synthase