THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 265, No. 28, Issue of October 5, pp. 17246-17251,199O 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S. A.

Molecular Cloning of the L-Amino-acid Oxidase Gene from Neurospora crassa*

(Received for publication, March 19, 1990)

Denise M. Niedermann and Konrad LerchS From the Biochemisches Institut der Uniuersitiit Ziirich, Winterthurerstrasse 190, Zlirich CH-8057, Switzerland

The addition of D-phenylalanine to starved cultures of Neurospora crassa leads to de novo synthesis of L- amino-acid oxidase. Poly(A) RNA from D-phenylala- nine-treated mycelium was therefore used to generate a cDNA library which was subsequently screened by hybrid-selected translation. A positive L-amino-acid oxidase clone served as a probe to isolate the complete gene from a genomic library of N. crassa. The nucleo- tide sequence obtained revealed an open reading frame coding for a protein of 695 amino acids. A compari- son of the deduced primary structure with the partial amino-terminal sequence of the isolated enzyme showed that the protein is synthesized as a precursor. The proform exceeds the mature enzyme by 129 amino acids. The presence of a cluster of basic amino acid residues preceding Ala12’ in the precursor suggests a post-translational modification brought about by lim- ited proteolysis. N. crassa L-amino-acid oxidase shares a highly conserved region with many well-character- ized flavoproteins that is known to constitute part of the flavin-adenine dinucleotide-binding site.

L-Amino-acid oxidase (EC 1.4.3.2) is a flavoenzyme which catalyzes the oxidative deamination of L-amino acids to the corresponding a-imino acids, which are then hydrolyzed to (Y- keto acids. In the course of the reaction, 2 electron eq are transferred from the amino acid to the flavin cofactor, which subsequently reduces molecular oxygen to hydrogen peroxide.

Re,H/NHl 0 II

+ 0, + H20 + R-C -COO- + NH: + H,O:!

Loo-

The enzyme is widely distributed in nature and has been purified from a number of different sources (Bright and Porter, 1975). The mechanism of L-amino-acid oxidase has been studied extensively (Massey and Gishla, 1983); however, very little is known in terms of its structural properties.

In the tilamentous fungus Neurospora crassa, L-amino-acid oxidase is synthesized in response to the addition of L-amino acids after nitrogen starvation (Sikora and Marzluf, 1982). In

* This work was supported by Swiss National Science Foundation Grants 3.236-0.85 and 3100-25351 and by the Kanton of Ziiricb. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) JO5621.

$ Present address: Givaudan Research Co., Ltd., Ueberlandstr. 138, Diibendorf CH-8600, Switzerland.

addition, the enzyme can also be induced by starvation in phosphate buffer and by the addition of protein synthesis inhibitors, D-amino acids, or ATP (Thayer and Horowitz, 1951; Horowitz, 1965; Prade and Terenzi, 1985). To arrive at a better understanding of the molecular structure and the regulation of biosynthesis of N. crassa L-amino-acid oxidase, we set out to clone its gene. In this paper, the isolation and characterization of the L-amino-acid oxidase gene are pre- sented. The possible characterization of the L-amino-acid oxidase gene is presented. The possible significance of the large amino-terminal extension is discussed.

MATERIALS AND METHODS’

RESULTS

Purification and Characterization of L-Amino-acid Oxi- dase-N. crassa L-amino-acid oxidase was purified from D

phenylalanine-induced cultures by a combination of ammo- nium sulfate precipitation, gel filtration on Sephacryl S-200, and DEAE-cellulose chromatography. The enzyme obtained after the last step had a specific activity of 45 units/mg and gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 1, he A). As shown in Fig. 2, purified L-amino-acid oxidase exhibited the typical absorption spec- trum of a flavoprotein with maxima at 465 and 380 nm. This enzyme preparation was subsequently used to raise polyclonal antibodies in rabbits and to generate a set of peptides for the positive identification of putative L-amino-acid oxidase clones (see below).

cDNA Cloning-A cDNA library was constructed using poly(A) RNA from cells highly induced for L-amino-acid oxidase synthesis. Double-stranded cDNA was tailed with dGTP and ligated into pUC8 previously linearized with Hind11 and tailed with dCTP. Escherichia coli JM83 cells were transformed, and the library was screened by hybrid- selected in uitro translation. Two positive clones (pD261 and pD264; Fig. 1, lane C) were found from a total of 400 colonies. The corresponding plasmid DNAs were isolated, yielding in- serts of - 800 base pairs each. cDNA clone pD261 was further characterized by restriction mapping and sequencing (Fig. 3A). Nucleotide sequence analysis of the 800-base pair frag- ment revealed an open reading frame coding for 220 amino acids. The open reading frame was followed by a TGA stop codon some 30 nucleotides from the 3’-terminal end. These data suggested that cDNA clone pD261 represents the 3’-

1 Portions of this paper (including “Materials and Methods,” Figs. 1-3 and 5, and Tables I and II) are presented in miniprint at the end of this paper. The abbreviations used are: LAO, L-amino-acid oxidase; SDS. sodium dodecvl sulfate: HPLC. high pressure liquid chromatop- raphi; PTH, pben$thiohyd&toin. Minipiint is easily read with tie aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

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Primary Structure of N. crassa L-Amino-acid Oxidase 17247

terminal coding part of the L-amino-acid oxidase gene. To confirm this hypothesis, purified L-amino-acid oxidase was digested with trypsin, and the resulting peptides were purified by gel filtration and high pressure liquid chromatography. Peptides D-l, C-l, and C-4 could be unambiguously positioned in the amino-terminal region of pD261, whereas peptide C-5 was found to represent the carboxyl-terminal end of L-amino- acid oxidase based on amino acid sequence analysis (Fig. 4).

Genomic Cloning-To obtain a full-length clone of L-amino- acid oxidase, a partial Sau3Al genomic library from N. crassa strain OR (Schechtman and Yanofsky, 1983) was screened by colony hybridization. As a probe, the nick-translated 375-base pair XhoI/SphI restriction fragment from pD261 was used. Three positive clones were isolated out of -20,000 screened colonies. Restriction enzyme and Southern analyses using the 375-base pair nick-translated fragment as a probe indicated that the 13.7-kilobase insert of pDLC2 contained the complete L-amino-acid oxidase gene. Of this insert, 3.1 kilobases were subjected to nucleotide sequence analysis following the strat- egy outlined in Fig. 3B. In general, every sequence was recon- firmed by a second analysis.

The transcription start site was determined by Sl nuclease mapping (Fig. 5). A single “P end-labeled restriction fragment from PDLCB (labeled at the PstI site at position +172 and ending at the ScrFI site at position -62) was hybridized to poly(A) RNA extracted from L-amino-acid oxidase-induced strain TS. Subsequent treatment with Sl nuclease left a major resistant fragment ending at Cyt” and several minor frag- ments between Gus+’ and Cyt+25.

Nucleotide Structure and Inferred Amino Acid Sequence- The nucleotide sequence of the L-amino-acid oxidase gene including the 5’- and 3’-flanking regions is shown in Fig. 4. The gene shows an open reading frame coding for a total of 695 amino acids. To confirm the amino terminus of L-amino- acid oxidase, the purified enzyme was carboxymethylated and subjected to automatic sequence analysis. Surprisingly, the amino acid sequence of the first 12 residues (Table II) did not agree with the sequence deduced from the DNA structure. A closer inspection of the data, however, revealed that the isolated enzyme represents a truncated form starting 129 residues past the initiator methionine (Fig. 4). These results are also in agreement with the molecular weights obtained by sodium dodecyl sulfate gel electrophoresis for the mature enzyme (64,000) and the in uitro translated polypeptide (75,000), as shown in Fig. 1. The deduced amino acid sequence of the mature form is further supported by amino acid com- positions and sequence data of several peptides (A-l, B-l, B- 2, C-2, and C-3; Fig. 4). Finally, the amino acid composition of isolated L-amino-acid oxidase agrees favorably with that calculated from the DNA sequence data (Table I).

DISCUSSION

The nucleotide sequence of the N. crassa L-amino-acid oxidase gene has been obtained from a partial cDNA and a complete genomic DNA clone. The gene from wild-type strain OR encodes a protein consisting of 695 amino acids (Fig. 4). Unlike most of the cloned genes from N. crassa, the L-amino- acid oxidase gene is devoid of introns (Gurr et al., 1987).

The determination of the transcription initiation site with Sl nuclease showed a major resistant band at position +l and several minor ones between positions +8 and +25 (Fig. 5). The major transcription initiation site is located at a distance of 122 bases upstream of the ATG translation start codon. From previously characterized nuclear N. crassa genes (Schechtmann and Yanofsky, 1983; Huiet and Giles, 1986), it was found that the length of the 5’-untranslated regions is

variable ranging from 28 up to 408 bases. The sequence CTACA of the major transcription start site in the L-amino- acid oxidase gene is similar to those of the ADP/ATP carrier gene (CTCCA; Arends and Sebald, 1984), the lactase gene (CTTCA, Germann et al., 1988), and the tyrosinase gene (CTACA, Kupper et al., 1989). The L-amino-acid oxidase gene contains a typical TATA box (Breathnach and Chambon, 1981) centered at position -33 (Fig. 4); the CAAT box, usually present in most eucaryotic promoters in the -100 region, however, is absent, as has been observed for several fungal genes (Gurr et al., 1987).

An inspection of the 3’-untranslated region of the L-amino- acid oxidase gene shows the absence of the typical eucaryotic poly(A) addition signal, AAUAAA (Proudfoot and Brownlee, 1976). A potential polyadenylation site (UUUUUAUAGG) particularly rich in thymidine residues is located at position +2382 (Fig. 4).

The guanine and cytosine content of the sequenced regions of the L-amino-acid oxidase gene is 54.8%, corresponding to the average value of 54% of the overall N. crassa genome (Villa and Storck, 1968). The guanine and cytosine content of the coding part (58.7%) is significantly enhanced compared to the 5’- and 3’-flanking regions (53.2 and 40.6%, respec- tively). This is due to the preference (77.9%) for codons ending in guanine or cytosine. Analysis of several N. crassa genes has led to the hypothesis that codon usage in this fungus correlates with the level of gene expression (Kinnaird and Fincham, 1983; Orbach et al., 1986). In genes which are subjected to strong regulation (Legerton and Yanofsky, 1985; Germann et al., 1988), nearly all possible triplets with a distinct preference for cytosine or guanine in the third posi- tion are represented. L-Amino-acid oxidase is an inducible enzyme; hence, its gene expression is expected to be under strong control. In agreement with the above-mentioned hy- pothesis, 59 out of 61 possible codons are used by the L- amino-acid oxidase gene.

The cloning and nucleotide sequence analysis of the N. crassa L-amino-acid oxidase gene revealed the rather surpris- ing result that the enzyme is synthesized as a precursor exceeding the mature form (566 amino acids) by 129 amino acids (Fig. 4). A comparison of the amino acid composition of the extension with the mature enzyme shows distinct differ- ences in the content of aromatic and basic amino acids. The native enzyme contains a total of 56 aromatic amino acids (10.3%), in contrast to only 7 residues (5.5%) in the amino- terminal extension. For the basic amino acids, the opposite is observed, with 20 residues (15.6%) found in the extension and only 46 (8.4%) in the mature form. Surprisingly, 16 of the 20 basic residues are clustered in the amino- and carboxyl- terminal ends of the extension (Fig. 4).

As is evident from automatic protein sequence analysis (Table II), the mature form starts at Ala+‘, which is located 129 residues from the initiator methionine. Ala” is preceded by a stretch of 5 basic amino acids (positions -1, -3, -6, -7, and -9), suggesting a proteolytic cleavage of the proenzyme by a protease with trypsin-like activity (Fig. 4). The biological significance of the rather long amino-terminal extension of N. crassa L-amino-acid oxidase is presently unknown. Under the induction conditions used in this study, L-amino-acid oxidase is present exclusively as an intracellular soluble en- zyme. Hence, it is rather unlikely that the amino-terminal extension could be involved in the secretion of the protein. This view is further supported by hydropathy analysis of the extension which did not indicate any characteristics of a secretory signal sequence. It is therefore most likely that proteolytic cleavage of the precursor could be somehow in-

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17248 Primary Structure of N. crassa L-Amino-acid Oxidase

SO nccTT!rz-&urclcc .

100 -C-mC&mTA

UtI.ysTx'p&rAla&gCly -129

1050 1100 cTcGcccccucT & &ccccmcrcuTcAcT~lTcrI~~ ~~~s~6sr-~~~~~ls~r~~n~~LyasarL~~c~yav~ V-r-PLYff

160 190 200 210

1150 TccKccC-&AcMcm&

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1550 1600 ccc-CcAm -T&l"2CTCD~CACCT2~ r ~roPhrS.rIl.Vr~y~Ly~T~Ph*&=P~~ LWlThr*lrP=0TtUL8- nA.laIlmClnUnL9-aclu~6uAla

350 ,-Cl-,/------------------------------------~-l--------------------------------

Cy~LY~VI~~CluPh~~~~T~l~~~L~roC~roIl~T~ly~~S= a-zhrmr&rAspIlrProclyIlrWp8 -----,,------c-,-------, 390 100 .lO

FIG. 4. Primary structure of N. crwsu L-amino-acid oxidase. Numbers refer to the nucleotide sequence and deduced amino acid sequence. Numbering begins at the major transcription initiation site and the amino terminus of mature L-amino-acid oxidase indicated by the arrow (see also text), respectively. The TATA sequence (Breathnach and Chambon, 1981) is bored. A putative polyadenylation site is underlined. The tryptic peptides isolated from mature L-amino-acid oxidase are indicated by dashed lines.

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LAO 48-77

DA0 2-31

MAO-A 15-43

I=". 100-128

GW. 22-50

PSBS 4-32

Primary Structure of N. crassa L-Amino-acid Oxidase

FIG. 6. Comparison of sequences involved in ADP-binding fold of L-amino-acid oxidase (LAO) and other flavoenzymes. DAO, n-amino-acid oxidase from pig (Fukui et al., 1987); MAO-A, monoamine oxidase from human (Hsu et al., 1988); MRase, mercuric reductase from Pseudomonas aeruginosa (Brown et al., 1983); GRuse, glutathione reductase from human (Krauth-Siegel et al., 1982); PHBH, p-hydroxybenzoate hydroxylase from Pseudomonas fluores- tens (Wierenga et al., 1983).

voived in enzyme activation. Alternatively, the processing of the proform may be simply the result of the presence of a proteolytically highly sensitive region in the enzyme.

A relatively large number of flavoenzymes have been stud- ied in terms of their primary and three-dimensional structures (Schirmer and Schultz, 1983). A comparison of the FAD- binding domains revealed a common particular &$&fold which binds the ADP moiety of the dinucleotide in an iden- tical manner (Wierenga et al., 1986). Moreover, this ADP- binding &$-fold was found to contain specific amino acid sequence features as shown in Fig. 6 for a number of flavoen- zymes. The predictive sequence fingerprint is the presence of a basic or hydrophilic residue in the first position, conserva- tive substitution of small hydrophobic residues in the first /3- fold (positions 2 and 4), as well as invariant glycine residues (positions 6, 8, and ll), small hydrophobic residues at posi- tions 15, 18, 25, and 27, and an acidic residue at position 29. For N. crassa L-amino-acid oxidase, the only divergence from this fingerprint is the substitution of a tyrosine residue at position 15. Interestingly, N. crassa L-amino-acid oxidase and pig D-amino-acid oxidase (Fukui et al., 1987) have an insertion of 1 amino acid between the end of the a-helix and the beginning of the P-strand (Fig. 6). As was pointed out by Wierenga et al. (1986), however, the length of the connecting loop (residues 22-24) can vary somewhat.

Acknowledgments-We thank U. Kupper for valuable discussions and Dr. M. G. Schechtmann for providing us with a genomic library.

17249

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Primary Structure of N. crassa L-Amino-acid Oxidase

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D M Niedermann and K LerchMolecular cloning of the L-amino-acid oxidase gene from Neurospora crassa.

1990, 265:17246-17251.J. Biol. Chem. 

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