Dissection of malonyl-Coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase
TL;DR: The results confirm Cys164's role as the active-site nucleophile in polyketide formation and elucidate the importance of His303 and Asn336 in the malonyl-CoA decarboxylation reaction.
Abstract: Chalcone synthase (CHS) catalyzes formation of the phenylpropanoid chalcone from one p-coumaroyl-CoA and three malonyl-coenzyme A (CoA) thioesters. The three-dimensional structure of CHS [Ferrer, J.-L., Jez, J. M., Bowman, M. E., Dixon, R. A., and Noel, J. P. (1999) Nat. Struct. Biol. 6, 775−784] suggests that four residues (Cys164, Phe215, His303, and Asn336) participate in the multiple decarboxylation and condensation reactions catalyzed by this enzyme. Here, we functionally characterize 16 point mutants of these residues for chalcone production, malonyl-CoA decarboxylation, and the ability to bind CoA and acetyl-CoA. Our results confirm Cys164's role as the active-site nucleophile in polyketide formation and elucidate the importance of His303 and Asn336 in the malonyl-CoA decarboxylation reaction. We suggest that Phe215 may help orient substrates at the active site during elongation of the polyketide intermediate. To better understand the structure−function relationships in some of these mutants, we al...
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1,474 citations
TL;DR: A major focus of this review is the integration of information from genetic and biochemical studies with the unique insights gained from protein X-ray crystallography and homology modeling, which generated a number of new predictions regarding both the importance and mechanistic role of various amino acid substitutions observed among functionally diverse type III PKS enzymes.
833 citations
TL;DR: Advances in four systems are reviewed: the cysteine synthase complex, the Calvin cycle, cyanogenic glucoside biosynthesis, and the phenylpropanoid pathway, providing new evidence for the importance of enzyme organization in cellular biochemistry as well as exclusive insights into the molecular basis of enzyme complex assembly.
Abstract: The organization of cooperating enzymes into macromolecular complexes is a central feature of cellular metabolism. A major advantage of such spatial organization is the transfer of biosynthetic intermediates between catalytic sites without diffusion into the bulk phase of the cell. This so-called "metabolic channeling" offers unique opportunities for enhancing and regulating cellular biochemistry. Studies in a number of plant primary and secondary metabolic systems continue to contribute to our understanding of the nature and importance of this phenomenon. This article reviews advances in four systems: the cysteine synthase complex, the Calvin cycle, cyanogenic glucoside biosynthesis, and the phenylpropanoid pathway. Each of these systems is providing new evidence for the importance of enzyme organization in cellular biochemistry as well as exclusive insights into the molecular basis of enzyme complex assembly. This review also explores current prospects for understanding metabolon structure, assembly, and biological function.
588 citations
TL;DR: Examples of polyketide synthases with much greater diversity in both mechanism and structure than the current type I, II and III paradigms serve as an inspiration for searching novel polyketid synthases to give new insights intopolyketide biosynthesis and to provide new opportunities for combinatorial biosynthesis.
474 citations
01 Jan 2007
TL;DR: The archetypical polyketide synthases (PKSs), known as type I, II, and III PKSs, have accounted for the vast structural diversities embodied by polyketides natural products, but recent progress in polyketid biosynthesis clearly suggests much greater diversity for PKS mechanism and structure as discussed by the authors.
Abstract: The archetypical polyketide synthases (PKSs), known as type I, II, and III PKSs, have accounted for the vast structural diversities embodied by polyketide natural products, but recent progress in polyketide biosynthesis clearly suggests much greater diversity for PKS mechanism and structure. We have previously argued the emergence of novel PKSs and cautioned the oversimplification of polyketide biosynthesis according to the type I, II, and III PKS paradigms on the basis of our studies on the biosynthesis of the enediynes, the macrotetrolides, and leinamycin. We present here a brief progress report on our current effort to mechanistically characterize these novel PKSs.
443 citations
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TL;DR: The PROCHECK suite of programs as mentioned in this paper provides a detailed check on the stereochemistry of a protein structure and provides an assessment of the overall quality of the structure as compared with well refined structures of the same resolution.
Abstract: The PROCHECK suite of programs provides a detailed check on the stereochemistry of a protein structure Its outputs comprise a number of plots in PostScript format and a comprehensive residue-by-residue listing These give an assessment of the overall quality of the structure as compared with well refined structures of the same resolution and also highlight regions that may need further investigation The PROCHECK programs are useful for assessing the quality not only of protein structures in the process of being solved but also of existing structures and of those being modelled on known structures
22,829 citations
TL;DR: The likelihood function for macromolecular structures is extended to include prior phase information and experimental standard uncertainties and the results derived are consistently better than those obtained from least-squares refinement.
Abstract: This paper reviews the mathematical basis of maximum likelihood The likelihood function for macromolecular structures is extended to include prior phase information and experimental standard uncertainties The assumption that different parts of a structure might have different errors is considered A method for estimating σA using `free' reflections is described and its effects analysed The derived equations have been implemented in the program REFMAC This has been tested on several proteins at different stages of refinement (bacterial α-amylase, cytochrome c′, cross-linked insulin and oligopeptide binding protein) The results derived using the maximum-likelihood residual are consistently better than those obtained from least-squares refinement
14,622 citations
TL;DR: The MOLSCRIPT program as discussed by the authors produces plots of protein structures using several different kinds of representations, including simple wire models, ball-and-stick models, CPK models and text labels.
Abstract: The MOLSCRIPT program produces plots of protein structures using several different kinds of representations. Schematic drawings, simple wire models, ball-and-stick models, CPK models and text labels can be mixed freely. The schematic drawings are shaded to improve the illusion of three dimensionality. A number of parameters affecting various aspects of the objects drawn can be changed by the user. The output from the program is in PostScript format.
13,971 citations
Book•
01 Jan 1977
TL;DR: The second edition of this biological reference aimed at undergraduates and graduates is as mentioned in this paper, which covers the structure and mechanism of enzymes, creating a guide to the current understanding of enzymology.
Abstract: This is the second edition of this biological reference aimed at undergraduates and graduates. The book covers the structure and mechanism of enzymes, creating a guide to the current understanding of enzymology.
3,231 citations