THESIS
2017
201 pages : illustrations (some color) ; 30 cm
Abstract
Vitamin K2, or menaquinone, is a broadly distributed lipophilic natural product that
plays an essential role as an electron carrier in the respiratory chain of a large number
of bacterial pathogens, such as Haemophilus influenzae, Mycobacterium tuberculosis,
and Staphylococcus aureus. This vitamin, is involved in various important biological
processes in mammals, such as blood coagulation and bone metabolism, its
biosynthetic pathway is absent in mammalian tissues, making its biosynthetic
enzymes in bacteria an attractive target for the development of novel antibiotics.
o-Succinylbenzoyl-CoA (OSB-CoA) synthetase, or MenE, is an essential
adenylate-forming enzyme that is responsible for the fifth commited step in the
classical menaquinone biosynthetic pathway. It catalyzes a two...[
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Vitamin K2, or menaquinone, is a broadly distributed lipophilic natural product that
plays an essential role as an electron carrier in the respiratory chain of a large number
of bacterial pathogens, such as Haemophilus influenzae, Mycobacterium tuberculosis,
and Staphylococcus aureus. This vitamin, is involved in various important biological
processes in mammals, such as blood coagulation and bone metabolism, its
biosynthetic pathway is absent in mammalian tissues, making its biosynthetic
enzymes in bacteria an attractive target for the development of novel antibiotics.
o-Succinylbenzoyl-CoA (OSB-CoA) synthetase, or MenE, is an essential
adenylate-forming enzyme that is responsible for the fifth commited step in the
classical menaquinone biosynthetic pathway. It catalyzes a two-step thioesterification
of OSB in which this carboxylate substrate is firstly activated by ATP to form an
OSB-AMP intermediate, followed by thioester formation with CoA in the 2nd half
reaction. Accordingly, MenE is structurally and functionally homologous to the
members from ANL superfamily, which includes the acyl/aryl-CoA synthetases, adenylation domains of nonribosomal peptide synthetases, and luciferases. These
enymes share the first half adenylation reaction and a common domain alternation
mechanism whereby they catalyze two partial reactions in two distinct conformations.
In order to gain the better understanding of the adenylation mechanism of the ANL
enzymes, we have solved the crystal structures of Bacillus subtilis MenE (bsMenE) in
a ligand-free form or in complex with two nucleotides ATP or AMP. On this basis, a
conserved pattern is identified in the interaction between ATP and other ANL
enzymes. It involves tight gripping interactions of the phosphate-binding loop (P-loop)
with the ATP triphosphate moiety and an open-closed conformational change to form
a compact adenylation active site. In MenE catalysis, this ATP-enzyme interaction
creates a new binding site for the carboxylate substrate, allowing revelation of the
determinants of substrate specificities and in-line alignment of the two substrates for
backside nucleophilic substitution reaction by molecular modeling. In addition, the
ATP-enzyme interaction is suggested to play a crucial catalytic role by mutation of
the P-loop residues hydrogen-bonded to ATP. Moreover, the ATP-enzyme interaction
has also clarified the positioning and catalytic role of a conserved lysine residue in
stabilization of the transition state. These findings provide new insights into the
adenylation half-reaction in the domain alteration catalytic mechanism of the
adenylate-forming enzymes.
Like many other adenylating enzymes, MenE undergoes a large C-domain rotation
to manage multi-substrates binding, catalysis, and product release in a dynamic
process. To further elucidate how the adenylation process occurs in the enzyme active
site, the crystal structure of its complex with the acyl-adenylate intermediate
OSB-AMP has been determind in a novel post-adenylation state at 2.69 Å resolution.
This structure presents unique features such as a strained conformation for the bound
adenylate intermediate to indicate that it represents the enzyme state after completion
of the adenylation reaction but before release of the C domain in its transition to the
thioesterification conformation. By comparison to the ATP-bound pre-adenylation conformation, structural changes are identified in both the reactants and the active site
to allow inference about how these changes accommodate and facilitate the
adenylation reaction and to directly support an in-line backside attack nucleophilic
substitution mechanism for the first half-reaction. Mutational analysis suggests that
the conserved His196 plays an important role in desolvation of the active site rather
than stabilizing the transition state of the adenylation reaction. In addition,
comparison of the new structure with a previously determined OSB-AMP-bound
structure of the same enzyme allows us to propose a release mechanism of the C
domain in its alteration to form the thioesterification conformation.
Finally, we have successfully determined the high resolution crystal structures of a
catalytically competent double mutant (IRAK) of bsMenE in the binary complex form
with a synthetic product analogue OSB-NCoA, or in ternary complex with both
OSB-NCoA and AMP. These structures captured at the 2nd partial reaction stage
reveals a 139.5° C-domain alternation which is likely to triggered by the third
substrate CoA. It is conceivable that both the enzyme and the coenzyme move
synergistically to achieve precise positioning of the nucleophilic CoA thiol deep into
the buried active site in the thioester-forming conformation. A crowbar-shaped
configuration of the OSB-NCoA plays an important role in stabilizing the
ligand-protein interaction. A large scale OSB succinyl group movement during the
2nd partial reaction underlies the thioester-forming mechanism using an activated
acyl-AMP as the reactant. Besides, a novel pantetheinyl tunnel, though constituted by
variable inter-domain residues, functions as a conserved and perfect director in
guiding the CoA sulfur to the well-protected adenylate intermediate. Lastly, two
distinct binding subsites for the CoA adenosine are identified on the surface of
N-domain or C-domain, which has shed new lights on the CoA binding affinity, the
conformational preference and the timing of the domain alternation throughout the
ANL enzymes. Taken together, it is the first time to provide the complete snapshots in
a single ANL enzyme for all the essential steps along the two-step catalysis, which
underlies the comprehensive structural basis of domain-alternation mechanism
ubiquitous to the adenylate-forming enzymes.
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