RESUMO
BX1 from Zea mays (zmBX1) is an enzyme of plant secondary metabolism that generates indole for the synthesis of plant defensins. It is a homologue of the tryptophan synthase α-subunit, TrpA. Whereas TrpA itself is a monomer in solution, zmBX1 is dimeric, confirmed in our work by native MS. Using cross-linking and mutagenesis, we identified the physiological dimerization interface of zmBX1. We found that homodimerization has only minor effects on catalysis and stability. A comparison of the zmBX1-zmBX1 homodimer and zmTrpA-zmTrpB heterodimer interfaces suggest that homodimerization in zmBX1 might, at an early point in evolution, have served as a mechanism to exclude the interaction with the tryptophan synthase ß-subunit (zmTrpB), marking its transition from primary to secondary metabolism.
Assuntos
Proteínas de Plantas/química , Triptofano Sintase/química , Zea mays/química , Sequência de Aminoácidos , Estabilidade Enzimática , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Espectrometria de Massas em TandemRESUMO
Tryptophan synthase (TS) is a heterotetrameric αßßα complex. It is characterized by the channeling of the reaction intermediate indole and the mutual activation of the α-subunit TrpA and the ß-subunit TrpB via a complex allosteric network. We have analyzed this allosteric network by means of ancestral sequence reconstruction (ASR), which is an in silico method to resurrect extinct ancestors of modern proteins. Previously, the sequences of TrpA and TrpB from the last bacterial common ancestor (LBCA) have been computed by means of ASR and characterized. LBCA-TS is similar to modern TS by forming a αßßα complex with indole channeling taking place. However, LBCA-TrpA allosterically decreases the activity of LBCA-TrpB, whereas, for example, the modern ncTrpA from Neptuniibacter caesariensis allosterically increases the activity of ncTrpB. To identify amino acid residues that are responsible for this inversion of the allosteric effect, all 6 evolutionary TrpA and TrpB intermediates that stepwise link LBCA-TS with ncTS were characterized. Remarkably, the switching from TrpB inhibition to TrpB activation by TrpA occurred between 2 successive TS intermediates. Sequence comparison of these 2 intermediates and iterative rounds of site-directed mutagenesis allowed us to identify 4 of 413 residues from TrpB that are crucial for its allosteric activation by TrpA. The effect of our mutational studies was rationalized by a community analysis based on molecular dynamics simulations. Our findings demonstrate that ancestral sequence reconstruction can efficiently identify residues contributing to allosteric signal propagation in multienzyme complexes.
Assuntos
Proteínas de Bactérias/genética , Biologia Computacional , Extinção Biológica , Subunidades Proteicas/genética , Triptofano Sintase/genética , Regulação Alostérica/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Oceanospirillaceae/genética , Oceanospirillaceae/metabolismo , Filogenia , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Triptofano/biossíntese , Triptofano Sintase/química , Triptofano Sintase/metabolismoRESUMO
The αßßα tryptophan synthase (TS), which is part of primary metabolism, is a paradigm for allosteric communication in multienzyme complexes. In particular, the intrinsically low catalytic activity of the α-subunit TrpA is stimulated several hundredfold through the interaction with the ß-subunit TrpB1. The BX1 protein from Zea mays (zmBX1), which is part of secondary metabolism, catalyzes the same reaction as that of its homologue TrpA, but with high activity in the absence of an interaction partner. The intrinsic activity of TrpA can be significantly increased through the exchange of several active-site loop residues, which mimic the corresponding loop in zmBX1. The subsequent identification of activating amino acids in the generated "stand-alone" TrpA contributes to an understanding of allostery in TS. Moreover, findings suggest an evolutionary trajectory that describes the transition from a primary metabolic enzyme regulated by an interaction partner to a self-reliant, stand-alone, secondary metabolic enzyme.
Assuntos
Complexos Multienzimáticos/metabolismo , Proteínas de Plantas/metabolismo , Triptofano Sintase/metabolismo , Zea mays/enzimologia , Sequência de Aminoácidos , Biocatálise , Evolução Biológica , Domínio Catalítico , Cinética , Modelos Moleculares , Complexos Multienzimáticos/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Homologia de Sequência de Aminoácidos , Triptofano Sintase/química , Triptofano Sintase/genética , Zea mays/genéticaRESUMO
Tryptophan synthase (TrpS) is a heterotetrameric αßßα enzyme that exhibits complex substrate channeling and allosteric mechanisms and is a model system in enzymology. In this work, we characterize proposed early and late evolutionary states of TrpS and show that they have distinct quaternary structures caused by insertions-deletions of sequence segments (indels) in the ß-subunit. Remarkably, indole hydrophobic channels that connect α and ß active sites have re-emerged in both TrpS types, yet they follow different paths through the ß-subunit fold. Also, both TrpS geometries activate the α-subunit through the rearrangement of loops flanking the active site. Our results link evolutionary sequence changes in the enzyme subunits with channeling and allostery in the TrpS enzymes. The findings demonstrate that indels allow protein quaternary architectures to escape "minima" in the evolutionary landscape, thereby overcoming the conservational constraints imposed by existing functional interfaces and being free to morph into new mechanistic enzymes.
