Cloning and sequencing of ATP sulfurylase from Penicillium chrysogenum. Identification of a likely allosteric domain.

B.A Foster,S.M Thomas,J.A Mahr,F Renosto,H.C Patel,I.H Segel

doi:10.1016/s0021-9258(17)32088-4

B.A Foster, S.M Thomas + Show 4 more

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https://doi.org/10.1016/s0021-9258(17)32088-4

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Abstract

Fungal (Penicillium chrysogenum) and yeast (Saccharomyces cerevisiae) ATP sulfurylases were shown to have very similar kinetic and chemical properties except that the fungal enzyme (a) contains a highly reactive Cys residue (SH-1) whose modification results in sigmoidal velocity curves (Renosto, F., Martin, R. L., and Segel, I. H. (1987) J. Biol. Chem. 262, 16279-16288) and (b) is allosterically inhibited by 3'-phosphoadenosine 5'-phosphosulfate (PAPS), while the yeast enzyme displays neither of these properties. The fungal enzyme subunit (64.3 kDa, 572 amino acids) is also larger than the yeast enzyme subunit (59.3 kDa, 521 amino acids). To correlate the unique allosteric properties of the fungal enzyme with specific structural features, we cloned and sequenced the ATP sulfurylase gene (aps) from P. chrysogenum. The yeast and fungal enzymes are homologous over the first 400 amino acids and contain two regions high in basic residues which are conserved in sulfurylases from Arabidopsis and the Riftia pachyptila (hydrothermal vent tube worm) chemolithotrophic symbiont. These regions may participate in forming the binding sites for MgATP2- and SO4(2-). The fungal enzyme has no sites for MgATP2- and SO4(2-). The fungal enzyme has no significant sequence homology to the yeast enzyme in the C-terminal 172 amino acids. This C-terminal region contains SH-1 (Cys-508) and has homology to MET14 (S. cerevisiae), CYSC (E. coli), and NODQ (Rhizobium meliloti), i.e. adenosine 5'-phosphosulfate (APS) kinase. The cumulative results suggest that (a) the allosteric PAPS binding site of P. chrysogenum ATP sulfurylase is located in the C-terminal domain of the protein and (b) that this domain may have evolved from APS kinase. In spite of the homology, this C-terminal region does not account for the APS kinase activity of P. chrysogenum. Fungal ATP sulfurylase has no significant homology to (or regulatory properties in common with) CYSD or CYSN, proteins reported to comprise E. coli ATP sulfurylase (Leyh, T., Vogt, T. F., and Suo, Y. (1992) J. Biol. Chem. 267, 10405-10410).

Highlights

From the Section of Molecular and CellularBiology, Division of Biological Sciences, University of California, Davis, California 95616
Genum is an oligomer composed of at least six identical subunits, each subunit possessinthgree free SH groups, is allosterically inhibited by 3’-phosphoadenosine5’phosphosulfate (PAPS), whilethe yeast enzyme displays neither of these properties
Estimates of the size of the fungal enzyme subunit (67-69 kDa) Sulfurylases-In spite of the kinetic similarities between the were based on results from minigels containing 1-5 pg of pro- yeast and fungal ATP sulfurylases, they respond quite differtein

Summary

AND DISCUSSION

Ceptible to inactivation by phenylglyoxal (an arginine-targeted reagent) and diethylpyrocarbonate Appears to be inactivated by this reagent onlyif activity is Both enzymes appear t o be composed of single-sized subunits measured a t subsaturating substrate levels [2] (because the with the fungal subunit (approximately 64 kDa) larger than main effect is on the [SI,, values rather than on hcnJ that of the yeast subunit (approximately 58 kDa). Kinetic Constants of theYeast ATP Sulfurylase-The kinetic by the fungal enzyme at K," levels of substrates is reduced to constants of the P. chrysogenum ATP sulfurylase are known This level of but, except for a few studies on labeled sulfate or selenate PAPS has abarely measurable effect on the yeast enzyme.

68 PM 192 PM 1040 PM

CONCLUSIONS

ATGGCCAACGCC

22 GAT Asp 8 GGT Gly 30 GAC Asp 28 GGC Gly