Oxytetracycline Biosynthesis

Oxytetracycline (OTC) is a broad-spectrum antibiotic that acts by inhibiting protein synthesis in bacteria. It is an important member of the bacterial aromatic polyketide family, which is a structurally diverse class of natural products. OTC is synthesized by a type II polyketide synthase that generates the poly-β-ketone backbone through successive decarboxylative condensation of malonyl-CoA extender units, followed by modifications by cyclases, oxygenases, transferases, and additional tailoring enzymes. Genetic and biochemical studies have illuminated most of the steps involved in the biosynthesis of OTC, which is detailed here as a representative case study in type II polyketide biosynthesis.     

Yet1p and Yet3p,the Yeast Homologs of BAP29 and BAP31, Interact with the Endoplasmic Reticulum Translocation Apparatus and Are Required for Inositol Prototrophy

The mammalian B-cell receptor-associated proteins of 29 and 31 kDa (BAP29 and BAP31) are conserved integral membrane proteins that have reported roles in endoplasmic reticulum (ER) quality control, ER export of secretory cargo, and programmed cell death. In this study we investigated the yeast homologs of BAP29 and BAP31, known as Yet1p and Yet3p, to gain insight on cellular function. We found that Yet1p forms a complex with Yet3p (Yet complex) and that complex assembly was important for subunit stability and proper ER localization. The Yet complex was not efficiently packaged into ER-derived COPII vesicles and therefore does not appear to act as an ER export receptor. Instead, a fraction of the Yet complex was detected in association with the ER translocation apparatus (Sec complex). Specific mutations in the Sec complex or Yet complex influenced these interactions. Moreover, associations between the Yet complex and Sec complex were increased by ER stress and diminished when protein translocation substrates were depleted. Surprisingly, yet1Δ and yet3Δ mutant strains displayed inositol starvation-related growth defects. In accord with the biochemical data, these growth defects were exacerbated by a combination of certain mutations in the Sec complex with yet1Δ or yet3Δ mutations. We propose a model for the Yet-Sec complex interaction that places Yet1p and Yet3p at the translocation pore to manage biogenesis of specific transmembrane secretory proteins.     

Glutamate 90 at the Luminal Ion Gate of Sarcoplasmic Reticulum Ca2+-ATPase Is Critical for Ca2+ Binding on Both Sides of the Membrane

The roles of Ser⁷², Glu⁹⁰, and Lys²⁹⁷ at the luminal ends of transmembrane helices M1, M2, and M4 of sarcoplasmic reticulum Ca²⁺-ATPase were examined by transient and steady-state kinetic analysis of mutants. The dependence on the luminal Ca²⁺ concentration of phosphorylation by P_i (“Ca²⁺ gradient-dependent E2P formation”) showed a reduction of the apparent affinity for luminal Ca²⁺ in mutants with alanine or leucine replacement of Glu⁹⁰, whereas arginine replacement of Glu⁹⁰ or Ser⁷² allowed E2P formation from P_i even at luminal Ca²⁺ concentrations much too small to support phosphorylation in wild type. The latter mutants further displayed a blocked dephosphorylation of E2P and an increased rate of conversion of the ADP-sensitive E1P phosphoenzyme intermediate to ADP-insensitive E2P as well as insensitivity of the E2·BeF₃⁻ complex to luminal Ca²⁺. Altogether, these findings, supported by structural modeling, indicate that the E2P intermediate is stabilized in the mutants with arginine replacement of Glu⁹⁰ or Ser⁷², because the positive charge of the arginine side chain mimics Ca²⁺ occupying a luminally exposed low affinity Ca²⁺ site of E2P, thus identifying an essential locus (a “leaving site”) on the luminal Ca²⁺ exit pathway. Mutants with alanine or leucine replacement of Glu⁹⁰ further displayed a marked slowing of the Ca²⁺ binding transition as well as slowing of the dissociation of Ca²⁺ from Ca₂E1 back toward the cytoplasm, thus demonstrating that Glu⁹⁰ is also critical for the function of the cytoplasmically exposed Ca²⁺ sites on the opposite side of the membrane relative to where Glu⁹⁰ is located.     

Identification of a Key Residue Determining Substrate Affinity in the Yeast Glucose Transporter Hxt7: A TWO-DIMENSIONAL COMPREHENSIVE STUDY*

We previously identified Asn³³¹ in transmembrane segment 7 (TM7) as a key residue determining substrate affinity in Hxt2, a moderately high-affinity facilitative glucose transporter of Saccharomyces cerevisiae. To gain further insight into the structural basis of substrate recognition by yeast glucose transporters, we have now studied Hxt7, whose affinity for glucose is the highest among the major hexose transporters. The functional role of Asp³⁴⁰ in Hxt7, the residue corresponding to Asn³³¹ of Hxt2, was examined by replacing it with each of the other 19 amino acids. Such replacement of Asp³⁴⁰ generated transporters with various affinities for glucose, with the affinity of the Cys³⁴⁰ mutant surpassing that of the wild-type Hxt7. To examine the structural role of Asp³⁴⁰ in the substrate translocation pathway, we performed cysteine-scanning mutagenesis of the 21 residues in TM7 of a functional Cys-less Hxt7 mutant in conjunction with exposure to the hydrophilic sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS). The transport activity of the D340C mutant of Cys-less Hxt7, in which Asp³⁴⁰ is replaced with Cys, was completely inhibited by pCMBS, indicating that Asp³⁴⁰ is located in a water-accessible position. This D340C mutant showed a sensitivity to pCMBS that was ∼70 times that of the wild-type Hxt7, and it was protected from pCMBS inhibition by the substrates d-glucose and 2-deoxy-d-glucose but not by l-glucose. These results indicate that Asp³⁴⁰ is situated at or close to a substrate recognition site and is a key residue determining high-affinity glucose transport by Hxt7, supporting the notion that yeast glucose transporters share a common mechanism for substrate recognition.     

Structure and Molecular Characterization of Streptococcus pneumoniae Capsular Polysaccharide 10F by Carbohydrate Engineering in Streptococcus oralis

Although closely related at the molecular level, the capsular polysaccharide (CPS) of serotype 10F Streptococcus pneumoniae and coaggregation receptor polysaccharide (RPS) of Streptococcus oralis C104 have distinct ecological roles. CPS prevents phagocytosis of pathogenic S. pneumoniae, whereas RPS of commensal S. oralis functions as a receptor for lectin-like adhesins on other members of the dental plaque biofilm community. Results from high resolution NMR identified the recognition region of S. oralis RPS (i.e. Galfβ1–6GalNAcβ1–3Galα) in the hexasaccharide repeat of S. pneumoniae CPS10F. The failure of this polysaccharide to support fimbriae-mediated adhesion of Actinomyces naeslundii was explained by the position of Galf, which occurred as a branch in CPS10F rather than within the linear polysaccharide chain, as in RPS. Carbohydrate engineering of S. oralis RPS with wzy from S. pneumoniae attributed formation of the Galf branch in CPS10F to the linkage of adjacent repeating units through sub terminal GalNAc in Galfβ1–6GalNAcβ1–3Galα rather than through terminal Galf, as in RPS. A gene (wcrD) from serotype 10A S. pneumoniae was then used to engineer a linear surface polysaccharide in S. oralis that was identical to RPS except for the presence of a β1–3 linkage between Galf and GalNAcβ1–3Galα. This polysaccharide also failed to support adhesion of A. naeslundii, thereby establishing the essential role of β1–6-linked Galf in recognition of adjacent GalNAcβ1–3Galα in wild-type RPS. These findings, which illustrate a molecular approach for relating bacterial polysaccharide structure to function, provide insight into the possible evolution of S. oralis RPS from S. pneumoniae CPS.     

Functional insights from the structural modelling of a small Fe-hydrogenase

Recently, a novel Fe-hydrogenase from a high rate of hydrogen producing Enterobacter cloacae strain IIT-BT08 was identified and partially characterized. This 147 residue protein was found to be much smaller than previously known Fe-hydrogenases, yet retaining a high catalytic activity. We predicted the structure of this protein and found it to be structurally similar to one of the two sub-domains containing the catalytic H-cluster so far jointly present in all other Fe-hydrogenases. This novel architecture allows a tentative explanation of protein function with the high rate of catalytic activity being due to a missing regulatory sub-domain, presumably allowing higher enzymatic activity at the cost of greater exposure to oxygen inactivation. This new insight may improve our understanding of the molecular and functional organization of other, more complex Fe-hydrogenases.     

LC-MS/MS based proteomic analysis and functional inference of hypothetical proteins in Desulfovibrio vulgaris

High efficiency capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to examine the proteins extracted from Desulfovibrio vulgaris cells across six treatment conditions. While our previous study provided a proteomic overview of the cellular metabolism based on proteins with known functions [W. Zhang, M.A. Gritsenko, R.J. Moore, D.E. Culley, L. Nie, K. Petritis, E.F. Strittmatter, D.G. Camp II, R.D. Smith, F.J. Brockman, A proteomic view of the metabolism in Desulfovibrio vulgaris determined by liquid chromatography coupled with tandem mass spectrometry, Proteomics 6 (2006) 4286-4299], this study describes the global detection and functional inference for hypothetical D. vulgaris proteins. Using criteria that a given peptide of a protein is identified from at least two out of three independent LC-MS/MS measurements and that for any protein at least two different peptides are identified among the three measurements, 129 open reading frames (ORFs) originally annotated as hypothetical proteins were found to encode expressed proteins. Functional inference for the conserved hypothetical proteins was performed by a combination of several non-homology based methods: genomic context analysis, phylogenomic profiling, and analysis of a combination of experimental information, including peptide detection in cells grown under specific culture conditions and cellular location of the proteins. Using this approach we were able to assign possible functions to 20 conserved hypothetical proteins. This study demonstrated that a combination of proteomics and bioinformatics methodologies can provide verification of the expression of hypothetical proteins and improve genome annotation.     

Presence of a vanadium-dependent haloperoxidase in Botrytis cinerea

The presence of a haloperoxidase in the mycelium of Botrytis cinerea, extractable with buffer, is demonstrated. A low level of extracellular enzyme activity was also detected. The haloperoxidase from the fungus is a vanadium-dependent glycoprotein, with a pH optimum of about 5.5. Native gel electrophoresis indicates that it is a high molecular mass protein. It appears to react with antibodies against haloperoxidase from Caldariomyces fumago. Enzyme activity is increased 3.5-fold and 15-fold by culture of the fungus in the presence of NaCl or vanadium, respectively. Activity is partly reduced by removal of vanadium and activity can be restored by the addition of vanadium to the enzyme. The possible function of this haloperoxidase is discussed.