xanthine oxidase, xanthine dehydrogenase, aldehyde oxidase, purine dehydrogenase, nitrate reductase, and formate dehy- drogenase, all of which require a ...
THEJOURNAL
OF
BIOLOGICAL CHEMISTRY
Val. 264, No. 35, Issue of December 15, pp. 20894-20901, 1989 Printed in U.S.A.
D 1989 by The American Society for Biochemistry and Molecular Biology, Inc.
Conserved Domains in Molybdenum Hydroxylases THE AMINO ACID SEQUENCEOFCHICKENHEPATICSULFITEOXIDASE* (Received for publication, July 10, 1989)
Peter J. NeameS and MichaelJ. Barber From the Departmentof Biochemistry and Molecular Biology, University of South Florida, College of Medicine and SShriners Hospital f a r Crippled Children, Tampa Unit, Tampa,Florida 33612
The amino acid sequence of the molybdenum-containing domain of chicken hepatic sulfite oxidase has been determined by Edman degradation of the purified protein. Combining these data with those previously published for the heme-containing domain (Guiard, B., and Lederer, F. (1979) Eur. J. Biochem. 100, 441453) indicates that each subunit of the homodimer comprises a single polypeptide chain containing 460 amino acid residues (Mr = 50,545). Comparison of the sequence with the cDNA-deduced sequence of assimilatory nitrate reductase from Arabidopsisthaliana shows a substantial degree of sequence conservation in the regions of the proteins that have been identified as comprising the Mo-pterin- and cytochrome b557-binding domains. These results suggest that the sequences forming the molybdenum-binding domains of the molybdenum hydroxylases may have evolvedfrom a common ancestral gene.
class of enzymes from a structural standpoint in that they consist of a variety of domains which have been rearranged genetically into different combinations to provide different functionality. While the NH2-terminal of sulfite oxidase is known to be in the class of b-type cytochromes, very little is known about the COOH-terminal, molybdenum-containing domain, although two characterized peptides from rat liver sulfite oxidase indicate that there issome homology with the equivalent domaininnitrate reductase (6). In contrast to sulfite oxidase,nitrate reductase has themolybdenum domain at the NH2 terminus of the molecule, the heme-containing domain in thecenter,andanadditionalflavin-containing domain at theCOOH terminus. Complete amino acid sequences, deduced from cDNA, have been obtained for theassimilatorynitrate reductasefrom Arabidopsis thaliana ( 6 )and tobacco (7) and also fromformate dehydrogenase from Methanobacteriumformicicum (8).There is extensive homology between the two plant reductases but no apparent relationship between nitrate reductase and the bacterial dehydrogenase. For nitrate reductase, which, in adSulfite oxidase (EC 1.8.3.1) catalyzes the terminal reaction dition to molybdenum, contains b-type cytochrome and FAD in the oxidative catabolism of the sulfur-containing amino prosthetic groups, significant homology has been found with acids, methionineand cysteine (1). The enzyme has been other b-type cytochrome-containing proteins and with the isolated from a variety of sources varying frombacteria (2) to flavoprotein, microsomal b5 reductase, suggesting that there mammals (3) and, with the exception of the monomeric bac- are conserved domains within the enzyme which correspond terial enzyme, has been shown to be ahomodimerwith to thepresence of specific cofactors. subunits of approximately 55 kDa, each of which contains a To date, detailed structural analysis of sulfite oxidase has Mo-pterinand b-typecytochrome (b557) prosthetic groups. beenconfined largely to the heme domain. This is readily Reducing equivalents from sulfite are donated to theenzyme isolated from the intact RL’ sulfite oxidase or chicken liver at the molybdenum center followed by intramolecular electron sulfite oxidase by tryptic (9) or chymotryptic (10) cleavage. transferto heme and egress to the physiological electron Thefragments which are derivedin this way havebeen characterized by size and composition. Based on SDS-PAGE, acceptor, cytochrome c (1). Sulfite oxidase is one of a group of enzymes referred to as the heme domain is 9.5 kDa while the molybdenum domain the “molybdenum hydroxylases” (4), the other enzymes being is 47 kDa (11).The composition of intact CL sulfite oxidase xanthine oxidase, xanthine dehydrogenase, aldehyde oxidase, (12) is similar to that of RL sulfite oxidase (11) (see Table M5). purine dehydrogenase, nitrate reductase, and formate dehyAmino acid sequence data have been obtained for the first drogenase, all of which require a molybdenum cofactor for catalytic activity. The structure of this cofactor has been 97 residues of the amino terminus of CL sulfite oxidase (10) Twotryptic established recently as a substituted pteringroup with molyb- that includes the b-typecytochromedomain. denum binding through two sulfur ligands andhas been peptides from the molybdenum domain of RL sulfite oxidase (6) have also been sequenced. Comparisonof these sequences termed “Mo-pterin” ( 5 ) . with the data obtainedfor A. thaliana nitrate reductase indiThe molybdenumhydroxylases representaninteresting cated that there may be an extensive degree of homology * This work was supported by Grants AR 35322 (to P. J. N.) and between the respective heme and molybdenum domains in GM 32696, a NationalInstitutes of Health BiomedicalResearch nitrate reductase and sulfite oxidase. Support Grant from the National Institutes of Health (to M. J. B.), To examine the extent of the homology between nitrate and a grant from the Shriners Hospitalsfor Crippled Children (to P. J. N.). The costs of publication of this article were defrayed in part reductase and sulfite oxidase molybdenum domains and to aid in identifying specific residues involved in binding to the by the paymentof page charges. This article must therefore hereby be marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact. The protein sequences(s) reported in this paper has been submitted with accession number(s) J05145. to the GenBankQ/EMBL Data Bank
The abbreviationsused are: SDS-PAGE,sodium dodecyl sulfatepolyacrylamide gel electrophoresis; DTT, dithiothreitol; CL, chicken liver; RL, rat liver; SOX, sulfite oxidase.
20894
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Sulfite Oxidase Mo-pterin prosthetic group, we have determined the amino acid sequence of the avian enzyme, CL sulfite oxidase. The sequence is strikingly similar to that of the molybdenum domain of plant nitrate reductase.
10
33
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40
5:
bC
WSYPRYTRPEVGRHRSFEE
RCWVTHGTDV FCVTDPiTLH P36rDKII.U XLhL:PBWR
i LYAVHGIPIN LZLLCOYKL"r
NTTAXMYSMS EVKXHNSA-D 550
SCWIIVHGHI YDCTEPLMDR PGGSDS1L:N AST--DCTEE 57c 580 590
FEAIHSUYXK -KMLELYRII, 6CC (10
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560
. .. . .. .
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90 .c0 110 120 130 140 ELSECFAZAA tDAQDEFiiGD P-PwPS-LkvT SQYSFNREPP.V.LLRERFLTPh€EFTRNHLPVPA'JEESS
150
. . . . .. .. .. . .. .. .. . . . .. .. . . . .. .. . . . .. .. . . . .. .. . . . .. ..
N A I L E P S V LDPRDEYT%DSWIERNPSW&W,:
MATERIALS AND METHODS AND RESULTS'
90
100
110
GKRPFNSEWLN"LLMI.HGFIT P'JPLHnTNH
: : . : . : . : : . : . : : 135
113
GWPKkG-WA
150
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