A Substrate-induced Biotin Binding Pocket in the Carboxyltransferase Domain of Pyruvate Carboxylase

Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the carboxyltransferase domain of PC from Rhizobium etli. Structures of the carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp590 and Tyr628 and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes

Insights Into the Carboxyltransferase Reaction of Pyruvate Carboxylase From the Structures of Bound Product and Intermediate Analogs

Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP- and bicarbonate-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. The carboxyltransferase (CT) domain of PC catalyzes the transfer of a carboxyl group from carboxybiotin to the accepting substrate, pyruvate. It has been hypothesized that the reactive enolpyruvate intermediate is stabilized through a bidentate interaction with the metal ion in the CT domain active site. Whereas bidentate ligands are commonly observed in enzymes catalyzing reactions proceeding through an enolpyruvate intermediate, no bidentate interaction has yet been observed in the CT domain of PC. Here, we report three X-ray crystal structures of the Rhizobium etli PC CT domain with the bound inhibitors oxalate, 3-hydroxypyruvate, and 3-bromopyruvate. Oxalate, a stereoelectronic mimic of the enolpyruvate intermediate, does not interact directly with the metal ion. Instead, oxalate is buried in a pocket formed by several positively charged amino acid residues and the metal ion. Furthermore, both 3-hydroxypyruvate and 3-bromopyruvate, analogs of the reaction product oxaloacetate, bind in an identical manner to oxalate suggesting that the substrate maintains its orientation in the active site throughout catalysis. Together, these structures indicate that the substrates, products and intermediates in the PC-catalyzed reaction are not oriented in the active site as previously assumed. The absence of a bidentate interaction with the active site metal appears to be a unique mechanistic feature among the small group of biotin-dependent enzymes that act on α-keto acid substrates

Functionally Diverse Biotin-dependent Enzymes with Oxaloacetate Decarboxylase Activity

Biotin-dependent enzymes catalyze carboxylation, decarboxylation and transcarboxylation reactions that participate in the primary metabolism of a wide range of organisms. In all cases, the overall reaction proceeds via two half reactions that take place in physically distinct active sites. In the first half-reaction, a carboxyl group is transferred to the 1-N′ of a covalently tethered biotin cofactor. The tethered carboxybiotin intermediate subsequently translocates to a second active site where the carboxyl group is either transferred to an acceptor substrate or, in some bacteria and archaea, is decarboxylated to biotin and CO2 in order to power the export of sodium ions from the cytoplasm. A homologous carboxyltransferase domain is found in three enzymes that catalyze diverse overall reactions: carbon fixation by pyruvate carboxylase, decarboxylation and sodium transport by the biotin-dependent oxaloacetate decarboxylase complex, and transcarboxylation by transcarboxylase from Propionibacterium shermanii. Over the past several years, structural data have emerged which have greatly advanced the mechanistic description of these enzymes. This review assembles a uniform description of the carboxyltransferase domain structure and catalytic mechanism from recent studies of pyruvate carboxylase, oxaloacetate decarboxylase and transcarboxylase, three enzymes that utilize an analogous carboxyltransferase domain to catalyze the biotin-dependent decarboxylation of oxaloacetate

The Role of Biotin and Oxamate in the Carboxyltransferase Reaction of Pyruvate Carboxylase

Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. During catalysis, carboxybiotin is translocated to the carboxyltransferase domain where the carboxyl group is transferred to the acceptor substrate, pyruvate. Many studies on the carboxyltransferase domain of PC have demonstrated an enhanced oxaloacetate decarboxylation activity in the presence of oxamate and it has been shown that oxamate accepts a carboxyl group from carboxybiotin during oxaloacetate decarboxylation. The X-ray crystal structure of the carboxyltransferase domain from Rhizobium etli PC reveals that oxamate is positioned in the active site in an identical manner to the substrate, pyruvate, and kinetic data are consistent with the oxamate-stimulated decarboxylation of oxaloacetate proceeding through a simple ping-pong bi bi mechanism in the absence of the biotin carboxylase domain. Additionally, analysis of truncated PC enzymes indicates that the BCCP domain devoid of biotin does not contribute directly to the enzymatic reaction and conclusively demonstrates a biotin-independent oxaloacetate decarboxylation activity in PC. These findings advance the description of catalysis in PC and can be extended to the study of related biotin-dependent enzymes

Structure of Mandelate Racemase with Bound Intermediate Analogues Benzohydroxamate and Cupferron

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg2+-dependent interconversion of the enantiomers of mandelate, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, we determined the X-ray crystal structures of wild-type MR complexed with two analogues of the putative aci-carboxylate intermediate, benzohydroxamate and Cupferron, to 2.2-Å resolution. Benzohydroxamate is shown to be a reasonable mimic of the transition state and/or intermediate because its binding affinity for 21 MR variants correlates well with changes in the free energy of transition state stabilization afforded by these variants. Both benzohydroxamate and Cupferron chelate the active site divalent metal ion and are bound in a conformation with the phenyl ring coplanar with the hydroxamate and diazeniumdiolate moieties, respectively. Structural overlays of MR complexed with benzohydroxamate, Cupferron, and the ground state analogue (S)-atrolactate reveal that the para carbon of the substrate phenyl ring moves by 0.8−1.2 Å between the ground state and intermediate state, consistent with the proposal that the phenyl ring moves during MR catalysis while the polar groups remain relatively fixed. Although the overall protein structure of MR with bound intermediate analogues is very similar to that of MR with bound (S)-atrolactate, the intermediate−Mg2+ distance becomes shorter, suggesting a tighter complex with the catalytic Mg2+. In addition, Tyr 54 moves closer to the phenyl ring of the bound intermediate analogues, contributing to an overall constriction of the active site cavity. However, site-directed mutagenesis experiments revealed that the role of Tyr 54 in MR catalysis is relatively minor, suggesting that alterations in enzyme structure that contribute to discrimination between the altered substrate in the transition state and the ground state by this proficient enzyme are extremely subtle

Potent Inhibition of Mandelate Racemase by a Fluorinated Substrate-Product Analogue with a Novel Binding Mode

Mandelate racemase (MR) from Pseudomonas putida catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Because trifluorolactate is also a substrate of MR, we anticipated that replacing the phenyl rings of the competitive, substrate-product analogue inhibitor benzilate (Ki = 0.7 mM) with trifluoromethyl groups might furnish an inhibitor. Surprisingly, the substrate-product analogue 3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)propanoate (TFHTP) was a potent competitive inhibitor [Ki = 27 ± 4 μM; cf. Km = 1.2 mM for both (R)-mandelate and (R)-trifluorolactate]. To understand the origins of this high binding affinity, we determined the X-ray crystal structure of the MR–TFHTP complex to 1.68 Å resolution. Rather than chelating the active site Mg2+ with its glycolate moiety, like other ground state analogues, TFHTP exhibited a novel binding mode with the two trifluoromethyl groups closely packed against the 20s loop and the carboxylate bridging the two active site Brønsted acid–base catalysts Lys 166 and His 297. Recognizing that positioning a carboxylate between the Brønsted acid–base catalysts could yield an inhibitor, we showed that tartronate was a competitive inhibitor of MR (Ki = 1.8 ± 0.1 mM). The X-ray crystal structure of the MR–tartronate complex (1.80 Å resolution) revealed that the glycolate moiety of tartronate chelated the Mg2+ and that the carboxylate bridged Lys 166 and His 297. Models of tartronate in monomers A and B of the crystal structure mimicked the binding orientations of (S)-mandelate and that anticipated for (R)-mandelate, respectively. For the latter monomer, the 20s loop appeared to be disordered, as it also did in the X-ray structure of the MR triple mutant (C92S/C264S/K166C) complexed with benzilate, which was determined to 1.89 Å resolution. These observations indicate that the 20s loop likely undergoes a significant conformational change upon binding (R)-mandelate. In general, our observations suggest that inhibitors of other enolase superfamily enzymes may be designed to capitalize on the recognition of the active site Brønsted acid–base catalysts as binding determinants

Diverse MarR bacterial regulators of auxin catabolism in the plant microbiome.

Chemical signalling in the plant microbiome can have drastic effects on microbial community structure, and on host growth and development. Previously, we demonstrated that the auxin metabolic signal interference performed by the bacterial genus Variovorax via an auxin degradation locus was essential for maintaining stereotypic root development in an ecologically relevant bacterial synthetic community. Here, we dissect the Variovorax auxin degradation locus to define the genes iadDE as necessary and sufficient for indole-3-acetic acid (IAA) degradation and signal interference. We determine the crystal structures and binding properties of the operon's MarR-family repressor with IAA and other auxins. Auxin degradation operons were identified across the bacterial tree of life and we define two distinct types on the basis of gene content and metabolic products: iac-like and iad-like. The structures of MarRs from representatives of each auxin degradation operon type establish that each has distinct IAA-binding pockets. Comparison of representative IAA-degrading strains from diverse bacterial genera colonizing Arabidopsis plants show that while all degrade IAA, only strains containing iad-like auxin-degrading operons interfere with auxin signalling in a complex synthetic community context. This suggests that iad-like operon-containing bacterial strains, including Variovorax species, play a key ecological role in modulating auxins in the plant microbiome