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Title: Structural and Kinetic Properties of Lumazine Synthase Isoenzymes in the Order Rhizobiales

Abstract

6, 7-Dimethyl-8-ribityllumazine synthase (lumazine synthase; LS) catalyzes the penultimate step in the biosynthesis of riboflavin in plants and microorganisms. This protein is known to exhibit different quaternary assemblies between species, existing as free pentamers, decamers (dimers of pentamers) and icosahedrally arranged dodecamers of pentamers. A phylogenetic analysis on eubacterial, fungal and plant LSs allowed us to classify them into two categories: Type I LSs (pentameric or icosahedral) and Type II LSs (decameric). The Rhizobiales represent an order of ?-proteobacteria that includes, among others, the genera Mesorhizobium, Agrobacterium and Brucella. Here, we present structural and kinetic studies on several LSs from Rhizobiales. Interestingly, Mesorhizobium and Brucella encode both a Type-I LS and a Type-II LS called RibH1 and RibH2, respectively. We show that Type II LSs appear to be almost inactive, whereas Type I LSs present a highly variable catalytic activity according to the genus. Additionally, we have solved four RibH1/RibH2 crystallographic structures from the genera Mesorhizobium and Brucella. The relationship between the active-site architecture and catalytic properties in these isoenzymes is discussed, and a model that describes the enzymatic behavior is proposed. Furthermore, sequence alignment studies allowed us to extend our results to the genus Agrobacterium. Our results suggest thatmore » the selective pressure controlling the riboflavin pathway favored the evolution of catalysts with low reaction rates, since the excess of flavins in the intracellular pool in Rhizobiales could act as a negative factor when these bacteria are exposed to oxidative or nitrosative stress.« less

Authors:
; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
959735
Report Number(s):
BNL-82721-2009-JA
Journal ID: ISSN 0022-2836; JMOBAK; TRN: US201016%%879
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Molecular Biology; Journal Volume: 373
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; ALIGNMENT; ARCHITECTURE; BACTERIA; BIOSYNTHESIS; BRUCELLA; CATALYSTS; DIMERS; ISOALLOXAZINES; ISOENZYMES; KINETICS; MICROORGANISMS; PROTEINS; REACTION KINETICS; RIBOFLAVIN; national synchrotron light source

Citation Formats

Klinke,S., Zylberman, V., Bonomi, H., Haase, I., Guimaraes, B., Braden, B., Bacher, A., Fischer, M., and Goldbaum, F. Structural and Kinetic Properties of Lumazine Synthase Isoenzymes in the Order Rhizobiales. United States: N. p., 2007. Web. doi:10.1016/j.jmb.2007.08.021.
Klinke,S., Zylberman, V., Bonomi, H., Haase, I., Guimaraes, B., Braden, B., Bacher, A., Fischer, M., & Goldbaum, F. Structural and Kinetic Properties of Lumazine Synthase Isoenzymes in the Order Rhizobiales. United States. doi:10.1016/j.jmb.2007.08.021.
Klinke,S., Zylberman, V., Bonomi, H., Haase, I., Guimaraes, B., Braden, B., Bacher, A., Fischer, M., and Goldbaum, F. Mon . "Structural and Kinetic Properties of Lumazine Synthase Isoenzymes in the Order Rhizobiales". United States. doi:10.1016/j.jmb.2007.08.021.
@article{osti_959735,
title = {Structural and Kinetic Properties of Lumazine Synthase Isoenzymes in the Order Rhizobiales},
author = {Klinke,S. and Zylberman, V. and Bonomi, H. and Haase, I. and Guimaraes, B. and Braden, B. and Bacher, A. and Fischer, M. and Goldbaum, F.},
abstractNote = {6, 7-Dimethyl-8-ribityllumazine synthase (lumazine synthase; LS) catalyzes the penultimate step in the biosynthesis of riboflavin in plants and microorganisms. This protein is known to exhibit different quaternary assemblies between species, existing as free pentamers, decamers (dimers of pentamers) and icosahedrally arranged dodecamers of pentamers. A phylogenetic analysis on eubacterial, fungal and plant LSs allowed us to classify them into two categories: Type I LSs (pentameric or icosahedral) and Type II LSs (decameric). The Rhizobiales represent an order of ?-proteobacteria that includes, among others, the genera Mesorhizobium, Agrobacterium and Brucella. Here, we present structural and kinetic studies on several LSs from Rhizobiales. Interestingly, Mesorhizobium and Brucella encode both a Type-I LS and a Type-II LS called RibH1 and RibH2, respectively. We show that Type II LSs appear to be almost inactive, whereas Type I LSs present a highly variable catalytic activity according to the genus. Additionally, we have solved four RibH1/RibH2 crystallographic structures from the genera Mesorhizobium and Brucella. The relationship between the active-site architecture and catalytic properties in these isoenzymes is discussed, and a model that describes the enzymatic behavior is proposed. Furthermore, sequence alignment studies allowed us to extend our results to the genus Agrobacterium. Our results suggest that the selective pressure controlling the riboflavin pathway favored the evolution of catalysts with low reaction rates, since the excess of flavins in the intracellular pool in Rhizobiales could act as a negative factor when these bacteria are exposed to oxidative or nitrosative stress.},
doi = {10.1016/j.jmb.2007.08.021},
journal = {Journal of Molecular Biology},
number = ,
volume = 373,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • Crystallographic studies of lumazine synthase, the penultimate enzyme of the riboflavin-biosynthetic pathway in B. anthracis, provide a structural framework for the design of antibiotic inhibitors, together with calorimetric and kinetic investigations of inhibitor binding. The crystal structure of lumazine synthase from Bacillus anthracis was solved by molecular replacement and refined to R{sub cryst} = 23.7% (R{sub free} = 28.4%) at a resolution of 3.5 Å. The structure reveals the icosahedral symmetry of the enzyme and specific features of the active site that are unique in comparison with previously determined orthologues. The application of isothermal titration calorimetry in combination with enzymemore » kinetics showed that three designed pyrimidine derivatives bind to lumazine synthase with micromolar dissociation constants and competitively inhibit the catalytic reaction. Structure-based modelling suggested the binding modes of the inhibitors in the active site and allowed an estimation of the possible contacts formed upon binding. The results provide a structural framework for the design of antibiotics active against B. anthracis.« less
  • Site-directed mutagenesis has been applied to improve the overexpression and purification of the icosahedral enzyme lumazine synthase from B. subtilis as well as to produce a new crystal form. The mutant protein crystallizes in space group R3 and diffracts X-rays to 1.6 Å resolution. Icosahedral macromolecules have a wide spectrum of potential nanotechnological applications, the success of which relies on the level of accuracy at which the molecular structure is known. Lumazine synthase from Bacillus subtilis forms a 150 Å icosahedral capsid consisting of 60 subunits and crystallizes in space group P6{sub 3}22 or C2. However, the quality of thesemore » crystals is poor and structural information is only available at 2.4 Å resolution. As classical strategies for growing better diffracting crystals have so far failed, protein engineering has been employed in order to improve the overexpression and purification of the molecule as well as to obtain new crystal forms. Two cysteines were replaced to bypass misfolding problems and a charged surface residue was replaced to force different molecular packings. The mutant protein crystallizes in space group R3, with unit-cell parameters a = b = 313.02, c = 365.77 Å, α = β = 90.0, γ = 120°, and diffracts to 1.6 Å resolution.« less
  • To increase the biochemical efficiency of biosynthetic systems, metabolic engineers have explored different approaches for organizing enzymes, including the generation of unnatural fusion proteins. Previous work aimed at improving the biosynthesis of resveratrol, a stilbene associated a range of health-promoting activities, in yeast used an unnatural engineered fusion protein of Arabidopsis thaliana (thale cress) 4-coumaroyl-CoA ligase (At4CL1) and Vitis vinifera (grape) stilbene synthase (VvSTS) to increase resveratrol levels 15-fold relative to yeast expressing the individual enzymes. Here we present the crystallographic and biochemical analysis of the 4CL::STS fusion protein. Determination of the X-ray crystal structure of 4CL::STS provides the firstmore » molecular view of an artificial didomain adenylation/ketosynthase fusion protein. Comparison of the steady-state kinetic properties of At4CL1, VvSTS, and 4CL::STS demonstrates that the fusion protein improves catalytic efficiency of either reaction less than 3-fold. Structural and kinetic analysis suggests that colocalization of the two enzyme active sites within 70 {angstrom} of each other provides the basis for enhanced in vivo synthesis of resveratrol.« less