RESUMO
The enzyme norcoclaurine synthase (NCS) catalyzes the stereospecific Pictet-Spengler cyclization between dopamine and 4-hydroxyphenylacetaldehyde, the key step in the benzylisoquinoline alkaloid biosynthetic pathway. The crystallographic structure of norcoclaurine synthase from Thalictrum flavum in its complex with dopamine substrate and the nonreactive substrate analogue 4-hydroxybenzaldehyde has been solved at 2.1A resolution. NCS shares no common features with the functionally correlated "Pictet-Spenglerases" that catalyze the first step of the indole alkaloids pathways and conforms to the overall fold of the Bet v1-like protein. The active site of NCS is located within a 20-A-long catalytic tunnel and is shaped by the side chains of a tyrosine, a lysine, an aspartic, and a glutamic acid. The geometry of the amino acid side chains with respect to the substrates reveals the structural determinants that govern the mechanism of the stereoselective Pictet-Spengler cyclization, thus establishing an excellent foundation for the understanding of the finer details of the catalytic process. Site-directed mutations of the relevant residues confirm the assignment based on crystallographic findings.
Assuntos
Alcaloides/biossíntese , Alcaloides/química , Carbono-Nitrogênio Ligases/química , Carbono-Nitrogênio Ligases/metabolismo , Tetra-Hidroisoquinolinas/química , Thalictrum/enzimologia , Biocatálise , Carbono-Nitrogênio Ligases/genética , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Thalictrum/genéticaRESUMO
Novel chimeric proteins made of a globin domain fused with a "cofactor free" monooxygenase domain have been identified within the Streptomyces avermitilis and Frankia sp. genomes by means of bioinformatics methods. Structure based sequence alignments show that the globin domains of both proteins can be unambiguously assigned to the truncated hemoglobin family, in view of the striking similarity to the truncated hemoglobins from Mycobacterium tuberculosis, Thermobifida fusca and Bacillus subtilis. In turn, the non-heme domains belong to a family of small (about 100 aminoacids) homodimeric proteins annotated as antibiotic biosynthesis monooxygenases, despite the lack of a cofactor (e.g., a metal, a flavin or a heme) necessary for oxygen activation. The chimeric protein from S. avermitilis has been cloned, expressed and characterized. The protein is a stable dimer in solution based on analytical ultracentrifugation experiments. The heme ligand binding properties with oxygen and carbonmonoxide resemble those of other Group II truncated hemoglobins. In addition, an oxygen dependent redox activity has been demonstrated towards easily oxidizable substrates such as menadiol and p-aminophenol. These findings suggest novel functional roles of truncated hemoglobins, which might represent a vast class of multipurpose oxygen activating/scavenging proteins whose catalytic action is mediated by the interaction with cofactor free monooxygenases.
Assuntos
Proteínas de Bactérias/genética , Hemeproteínas/genética , Oxigenases de Função Mista/genética , Proteínas Recombinantes de Fusão/genética , Streptomyces/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Monóxido de Carbono/metabolismo , Dimerização , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Oxirredutases/metabolismo , Oxigênio/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Espectrofotometria Ultravioleta , Streptomyces/metabolismoRESUMO
Transcription of the catabolic touABCDEF operon, encoding the toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1, is driven by the sigma(54)-dependent Ptou promoter, whose activity is controlled by the phenol-responsive NtrC-like activator TouR. In this paper we describe for the first time a peculiar characteristic of this system, namely, that Ptou transcription is activated in a growth phase-dependent manner in the absence of genuine effectors of the cognate TouR regulator. This phenomenon, which we named gratuitous activation, was observed in the native strain P. stutzeri OX1, as well as in a Pseudomonas putida PaW340 host harboring the reconstructed tou regulatory circuit. Regulator-promoter swapping experiments demonstrated that the presence of TouR is necessary and sufficient for imposing gratuitous activation on the Ptou promoter, as well as on other sigma(54)-dependent catabolic promoters, whereas the highly similar phenol-responsive activator DmpR is unable to activate the Ptou promoter in the absence of effectors. We show that this phenomenon is specifically triggered by carbon source exhaustion but not by nitrogen starvation. An updated model of the tou regulatory circuit is presented.
