A Protein Pre-Organized to Trap the Nucleotide Moiety of Coenzyme B12: Refined Solution Structure of the B12-Binding Subunit of Glutamate Mutase from Clostridium tetanomorphum
✍ Scribed by Bernd Hoffmann; Martin Tollinger; Robert Konrat; Marja Huhta; E. Neil G. Marsh; Bernhard Kräutler
- Publisher
- John Wiley and Sons
- Year
- 2001
- Tongue
- English
- Weight
- 476 KB
- Volume
- 2
- Category
- Article
- ISSN
- 1439-4227
No coin nor oath required. For personal study only.
✦ Synopsis
Dedicated to Professor Wolfgang Buckel on the occasion of his 60th birthday Uniformly 13 C, 15 N-labeled MutS, the coenzyme B 12 -binding subunit of glutamate mutase from Clostridium tetanomorphum, was prepared by overexpression from an Escherichia coli strain. Multidimensional heteronuclear NMR spectroscopic experiments with aqueous solutions of 13 C, 15 N-labeled MutS provided signal assignments for roughly 90 % of the 1025 hydrogen, 651 carbon, and 173 nitrogen atoms and resulted in about 1800 experimental restraints. Based on the information from the NMR experiments, the structure of MutS was calculated, confirming the earlier, less detailed structure obtained with 15 N-labeled MutS. The refined analysis allowed a precise determination of the secondary and tertiary structure including several crucial side chain interactions. The structures of (the apoprotein) MutS in solution and of the B 12binding subunit in the crystal of the corresponding homologous holoenzyme from Clostridium cochlearium differ only in a section that forms the well-structured helix a1 in the crystal structure and that also comprises the cobalt-coordinating histidine residue. In the apoprotein MutS, this part of the B 12 -binding subunit is dynamic. The carboxy-terminal end of this section is conformationally flexible and has significant propensity for an a-helical structure (ªnascent helixº). This dynamic section in MutS is a decisive element for the binding of the nucleotide moiety of coenzyme B 12 and appears to be stabilized as a helix (a1) upon trapping of the nucleotide of the B 12 cofactor.
KEYWORDS:
coenzyme B 12 ´glutamate mutase ´isomerases ŃMR spectroscopy ´protein structures Glutamate mutase (Glm) from Clostridium tetanomorphum [1] and Clostridium cochlearium [2] depends upon an adenosyl-cobamide cofactor and catalyzes the reversible carbon skeleton rearrangement of (S)-glutamate to (2S,3S)-3-methylaspartate (Scheme 1). This isomerization is the first step in the fermentation of glutamate to ammonia, CO 2 , acetate, butyrate, and H 2 by these bacteria. [3,4] Glm is composed of two components: E, a homodimer (e 2 , M r 107 600 Da) and S, a monomer (s, M r 14 700 Da). [2,5] The active Glm holoenzyme is an e 2 s 2 heterotetramer [6] which binds two molecules of coenzyme B 12 . [7,8] The genes coding for the e and s chains have been cloned from both clostridia in Escherichia coli. They were designated as mut genes in C. tetanomorphum [9,10] and as glm genes in C. cochlearium. [11] The mutS/mutE genes code for the subunits MutS and MutE of Glm from C. tetanomorphum, while the glmS/glmE genes code for the corresponding GlmS and GlmE subunits from C. cochlearium. MutS and GlmS show 84 % identity and were identified as the Glm B 12 -binding subunits, based on their deduced amino acid sequences [6,9,12] (Figure 1). The substrate-binding subunits MutE and GlmE are even 90 % identical, but no significant similarity to any other proteins is known. An X-ray crystallographic analysis of reconstituted Glm from
[a] Prof.