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Structural environment of an essential cysteine residue of xylanase from Chainia sp. (NCL 82.5.1).

ABSTRACT: N-(2,4-Dinitroanilino)maleimide (DAM) reacts covalently with the thiol group of the xylanase from Chainia leading to complete inactivation in a manner similar to N-ethylmaleimide, but provides a reporter group at the active site of the enzyme. Increasing amounts of xylan offered enhanced protection against inactivation of the xylanase by DAM. Xylan (5 mg) showed complete protection, providing evidence for the presence of cysteine at the substrate-binding site of the enzyme. Kinetics of chemical modification of the xylanase by DAM indicated the involvement of 1 mol of cysteine residue per mol of enzyme, as reported earlier [Deshpande, Hinge and Rao (1990) Biochim. Biophys. Acta 1041, 172-177]. The second-order rate constant for the reaction of DAM with the enzyme was 3.61 x 10(3) M-1.min-1. The purified xylanase was alkylated with DAM and digested with pepsin. The peptides were separated by gel filtration. The specific modified cysteinyl peptide was further purified by reverse-phase HPLC. The active-site peptide was located visually by its predominant yellow colour and characterized by a higher A340 to A210 ratio. The modified active-site peptide has the sequence: Glu-Thr-Phe-Xaa-Asp. The sequence of the peptide was distinctly different from that of cysteinyl peptide derived from a xylanase from a thermotolerant Streptomyces species, but showed the presence of a conserved aspartic acid residue consistent with the catalytic regions of other glucanases.

SUBMITTER: Rao M

PROVIDER: S-EPMC1217417 | biostudies-other | 1996 Jun

REPOSITORIES: biostudies-other

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Project description:BackgroundThiamine (vitamin B1) is synthesized de novo by certain yeast, fungi, plants, protozoans, bacteria and archaea. The pathway of thiamine biosynthesis by archaea is poorly understood, particularly the route of sulfur relay to form the thiazole ring. Archaea harbor structural homologs of both the bacterial (ThiS-ThiF) and eukaryotic (THI4) proteins that mobilize sulfur to thiazole ring precursors by distinct mechanisms.ResultsBased on comparative genome analysis, halophilic archaea are predicted to synthesize the pyrimidine moiety of thiamine by the bacterial pathway, initially suggesting that also a bacterial ThiS-ThiF type mechanism for synthesis of the thiazole ring is used in which the sulfur carrier ThiS is first activated by ThiF-catalyzed adenylation. The only ThiF homolog of Haloferax volcanii (UbaA) was deleted but this had no effect on growth in the absence of thiamine. Usage of the eukaryotic THI4-type sulfur relay was initially considered less likely for thiamine biosynthesis in archaea, since the active-site cysteine residue of yeast THI4p that donates the sulfur to the thiazole ring by a suicide mechanism is replaced by a histidine residue in many archaeal THI4 homologs and these are described as D-ribose-1,5-bisphosphate isomerases. The THI4 homolog of the halophilic archaea, including Hfx. volcanii (HVO_0665, HvThi4) was found to differ from that of methanogens and thermococci by having a cysteine residue (Cys165) corresponding to the conserved active site cysteine of yeast THI4p (Cys205). Deletion of HVO_0665 generated a thiamine auxotroph that was trans-complemented by a wild-type copy of HVO_0665, but not the modified gene encoding an HvThi4 C165A variant.ConclusionsBased on our results, we conclude that the archaeon Hfx. volcanii uses a yeast THI4-type mechanism for sulfur relay to form the thiazole ring of thiamine. We extend this finding to a relatively large group of archaea, including haloarchaea, ammonium oxidizing archaea, and some methanogen and Pyrococcus species, by observing that these organisms code for THI4 homologs that have a conserved active site cysteine residue which is likely used in thiamine biosynthesis. Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis.

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Structural environment of an essential cysteine residue of xylanase from Chainia sp. (NCL 82.5.1).

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