Structural and mechanistic investigation of the domain alternation catalysis of an adenylating enzyome (MenE) in vitamin K biosynthesis

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-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.

Date of Award	2017
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology