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Importance of tyrosine residues of Bacillus stearothermophilus serine hydroxymethyltransferase in cofactor binding and L-allo-Thr cleavage Crystal structure and biochemical studies

Bhavani, B. S. and Rajaram, V. and Shveta, Bisht and Purnima Kaul, Tiku and Prakash, V. (2008) Importance of tyrosine residues of Bacillus stearothermophilus serine hydroxymethyltransferase in cofactor binding and L-allo-Thr cleavage Crystal structure and biochemical studies. FEBS Journal, 275. pp. 4606-4619.

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Abstract

Serine hydroxymethyltransferase (SHMT) from Bacillus stearothermophilus (bsSHMT) is a pyridoxal 5¢-phosphate-dependent enzyme that catalyses the conversion of l-serine and tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. In addition, the enzyme catalyses the tetrahydrofolateindependent cleavage of 3-hydroxy amino acids and transamination. In this article, we have examined the mechanism of the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids by SHMT. The three-dimensional structure and biochemical properties of Y51F and Y61A bsSHMTs and their complexes with substrates, especially l-allo-Thr, show that the cleavage of 3-hydroxy amino acids could proceed via Ca proton abstraction rather than hydroxyl proton removal. Both mutations result in a complete loss of tetrahydrofolate-dependent and tetrahydrofolate-independent activities. The mutation of Y51 to F strongly affects the binding of pyridoxal 5¢-phosphate, possibly as a consequence of a change in the orientation of the phenyl ring in Y51F bsSHMT. The mutant enzyme could be completely reconstituted with pyridoxal 5¢-phosphate. However, there was an alteration in the kmax value of the internal aldimine (396 nm), a decrease in the rate of reduction with NaCNBH3 and a loss of the intermediate in the interaction with methoxyamine (MA). The mutation of Y61 to A results in the loss of interaction with Ca and Cb of the substrates. X-Ray structure and visible CD studies show that the mutant is capable of forming an external aldimine. However, the formation of the quinonoid intermediate is hindered. It is suggested that Y61 is involved in the abstraction of the Ca proton from 3-hydroxy amino acids. A new mechanism for the cleavage of 3-hydroxy amino acids via Ca proton abstraction by SHMT is proposed.

Item Type: Article
Uncontrolled Keywords: crystal structure; proton abstraction; pyridoxal 5¢-phosphate-dependent enzymes; serine hydroxymethyltransferase; tetrahydrofolate-independent cleavage
Subjects: 500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 16 Enzyme Chemistry
Divisions: Protein Chemistry and Technology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 02 Jan 2009 09:40
Last Modified: 01 Jun 2012 11:35
URI: http://ir.cftri.com/id/eprint/8859

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