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Functional analysis of active amino acid residues of the mercaptosuccinate dioxygenase of Variovorax paradoxus B4.

Abstract：

:Thiol dioxygenases are non-heme mononuclear-iron proteins and belong to the cupin superfamily. In 2014, mercaptosuccinate dioxygenase (Msdo) of Variovorax paradoxus B4 was identified as another bacterial cysteine dioxygenase (Cdo) homolog catalyzing the conversion of mercaptosuccinate (MS) into succinate and sulfite. To gain further insights into potentially important amino acid residues for enzyme activity, seven enzyme variants were generated and analyzed. (i) Three variants comprised the substitution of one conserved histidine residue each by leucine, either supposed to be mandatory for coordination of the Fe(II) cofactor (H93 and H95) or to be important for substrate positioning within the active site (H163). The corresponding enzyme variants were completely inactive confirming their essential roles for enzyme activity. (ii) Mutation C100S resulted as well in an inactive enzyme demonstrating its importance for either stability or activity of the protein. (iii) For eukaryotic Cdo, a hydrogen bond network for substrate positioning was postulated, and the corresponding amino acids are basically present in Msdo. Albeit the MsdoQ64A mutation exhibited an increased Km of 0.29 mM when compared to the wildtype with 0.06 mM, it did not significantly affect the specific activity. (iv) The variant MsdoR66A showed only very low activity even when high amounts of enzyme were applied indicating that this residue might be important for catalysis. (v) No strong effect had the mutation Y165F for which a specific enzyme activity of 10.22 μmol min-1 mg-1 protein and a Km value of 0.06 mM with high similarity to those of the wildtype enzyme were obtained. This residue corresponds to Y157 of human Cdo, which is part of the catalytic triad and is supposed to be involved in substrate positioning. Apparently, another residue could fulfill this role in Msdo, since the loss of Y165 did not have a strong effect.

journal_name

Enzyme Microb Technol

journal_title

Enzyme and microbial technology

authors

Brandt U,Galant G,Meinert-Berning C,Steinbüchel A

doi

10.1016/j.enzmictec.2018.09.007

subject

Has Abstract

pub_date

2019-01-01 00:00:00

pages

61-68

eissn

0141-0229

issn

1879-0909

pii

S0141-0229(18)30316-8

journal_volume

120

pub_type

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Functional analysis of active amino acid residues of the mercaptosuccinate dioxygenase of Variovorax paradoxus B4.