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Organization of the Multiple Coenzymes and Subunits and Role of the Covalent Flavin Link in the Complex Heterotetrameric Sarcosine Oxidase
Journal article   Peer reviewed

Organization of the Multiple Coenzymes and Subunits and Role of the Covalent Flavin Link in the Complex Heterotetrameric Sarcosine Oxidase

Michel Eschenbrenner, Lawrence J Chlumsky, Peeyush Khanna, Francoise Strasser and Marilyn Schuman Jorns
Biochemistry (Easton), v 40(18), pp 5352-5367
08 May 2001
DOI: https://doi.org/10.1021/bi010101p
PMID: 11330998
Featured in Collection :   UN Sustainable Development Goals @ Drexel

Abstract

Heterotetrameric (αβγδ) sarcosine oxidase from Corynebacterium sp. P-1 (cTSOX) contains noncovalently bound FAD and NAD+ and covalently bound FMN, attached to β(His173). The β(His173Asn) mutant is expressed as a catalytically inactive, labile heterotetramer. The β and δ subunits are lost during mutant enzyme purification, which yields a stable αγ complex. Addition of stabilizing agents prevents loss of the δ but not the β subunit. The covalent flavin link is clearly a critical structural element and essential for TSOX activity or preventing FMN loss. The α subunit was expressed by itself and purified by affinity chromatography. The α and β subunits each contain an NH2-terminal ADP-binding motif that could serve as part of the binding site for NAD+ or FAD. The α subunit and the αγ complex were each found to contain 1 mol of NAD+ but no FAD. Since NAD+ binds to α, FAD probably binds to β. The latter could not be directly demonstrated since it was not possible to express β by itself. However, FAD in TSOX from Pseudomonas maltophilia (pTSOX) exhibits properties similar to those observed for the covalently bound FAD in monomeric sarcosine oxidase and N-methyltryptophan oxidase, enzymes that exhibit sequence homology with β. A highly conserved glycine in the ADP-binding motif of the α(Gly139) or β(Gly30) subunit was mutated in an attempt to generate NAD+- or FAD-free cTSOX, respectively. The α(Gly139Ala) mutant is expressed only at low temperature (t optimum = 15 °C), but the purified enzyme exhibited properties indistinguishable from the wild-type enzyme. The much larger barrier to NAD+ binding in the case of the α(Gly139Val) mutant could not be overcome even by growth at 3 °C, suggesting that NAD+ binding is required for TSOX expression. The β(Gly30Ala) mutant exhibited subunit expression levels similar to those of the wild-type enzyme, but the mutation blocked subunit assembly and covalent attachment of FMN, suggesting that both processes require a conformational change in β that is induced upon FAD binding. About half of the covalent FMN in recombinant preparations of cTSOX or pTSOX is present as a reversible covalent 4a-adduct with a cysteine residue. Adduct formation is not prevented by mutating any of the three cysteine residues in the β subunit of cTSOX to Ser or Ala. Since FMN is attached via its 8-methyl group to the β subunit, the FMN ring must be located at the interface between β and another subunit that contains the reactive cysteine residue.

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Web of Science research areas
Biochemistry & Molecular Biology
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