Cloning and nucleotide sequencing of phenylalanine dehydrogenase gene of Bacillus sphaericus

337

Gene, 63 (1988) 337-341 Elsevier GEN 02315

Cloning

and nucleotide

(Activity

staining;

promoter;

sequencing

recombinant

of phenylalanine

DNA;

restriction

map;

dehydrogenase Southern-blot

gene of Bacillus sphaericus analysis;

translational

initiation

site;

terminator)

Noriko Okazaki, Yasuo Hibino, Yasubisa Asano, Muneki Obmori, Naganori Numao and Kiyosi Kondo Sagami

Chemical

Research

Received

21 August

Accepted

10 October

Received

by publisher

Center, Nishi-Ohnuma.

Sagamihara,

Kanagawa

229 (Japan)

1987 1987 16 December

1987

SUMMARY

The gene coding for phenylalanine dehydrogenase [ PDH; L-phenylalanine : NAD + oxidoreductase (deaminating); EC 1.4.1.-l from BacilZus sphaeticus SCRC-79a was cloned onto plasmid pUC9, and the nucleotide sequence of the 2-kb DNA region of the insert was determined. A 1143-bp open reading frame consisting of 381 codons was identified as a pdh gene coding for PDH.

INTRODUCTION

alanine. The enzyme appears to be useful as an industrial catalyst in the asymmetric synthesis of

NAD+ -dependent PDH (EC 1.4.1.-) catalyzes the reversible oxidative deamination of L-phenyl-

L-phenylalanine and related L-amino acids from their oxo-analogs. PDH was first discovered by Hummel et al. (1984) in Brevibacterium sp. Recently, Asano et al. (1987) found the enzyme activity in other bacterial species, B. sphaericus and Sporosurcinu ureue, and extensively investigated the physical, catalytic, and immunological properties of the enzymes from these organisms. While the Sporosurcinu enzyme showed high substrate specihcity toward L-phenylalanine, the enzyme from B. sphuericus acted on L-phenylalanine and L-tyrosine. Here we report the cloning and sequencing of the pdh gene from B. sphuericus.

Correspondence to: Dr. Y. Asano, Center,

Nishi-Ohnuma

Japan,

Tel. (0427)42-479

Abbreviations: coding

for

4-4-1,

aa, amino

PMS,

phenazine dodecyl

0378-I 119/88/$03.50

0

229,

pair(s);

bla, gene

high-performance

liquid

bp, base

kb, kilobase

nt, nucleotide(s);

frame; PDH, phenylalanine SDS, sodium

Research

Kanagawa

2(p-iodophenyl)-3-(p-nitrophenyl)-5-

chloride;

lac promoter-operator; PDH;

acid(s); HPLC,

INT,

phenyltetrazolium

Chemical

1

/l-lactamase;

chromatography;

Sagami Sagamihara,

dehydrogenase; methosulfate;

or 1000 bp; IacZpo, ORF, SD,

reading

Shine-Dalgarno;

sulfate.

1988 Elsevier

open

pdh, gene coding for

Science Publishers

B.V. (Biomedical Division)

338

EXPERIMENTAL

incubated

AND DISCUSSION

(a) Cloning of the @/I gene

0.2 M Tris * HCl pH 8.5, 1.5 mM NAD’,

alanine, Chromosomal DNA from B. sphaericus was partially digested with restriction endonuclease HindIII,

0.32 mM PMS, and 0.8 mM INT) and incubated room temperature

ligated into the Hind111 site of pUC9 plasmid (Vieira and Messing, 1982), and the mixture was used to transform were staining

for

the

and the PDH as described

expression enzyme

of ampicillin

The 5’-terminal

et al., 1986).

colonies

A colony pro-

colony was 14 kb in size and designated pBPDH1. Its restriction map is shown in Fig. la and b.

activity by colony

below (Inagaki

Briefly, the ampicillin-resistant

for a few minutes.

at

ducing PDH became dark red. Of about 5000 colonies screened, a single positive colony was identified. The plasmid DNA isolated from the

Escherichiu coli JM103. The transformants

screened

resistance

at 55°C for 10 min. The filter was trans-

ferred to a dish containing 0.5 ml of a reaction mixture for activity staining (10 mM L-phenyl-

region of the coding sequence

the pdh gene was localized

grown on a

by using

of

a synthetic

32P-end-labeled mixed probe [ 5’-dGC(A/G/T)AT(T/C)T(T/C)T(T/C)TG(A/G)AA-3’1 consisting

nitrocellulose filter were lysed, frozen at -7O”C, thawed at room temperature, and then the filter was

(a)

b) 51 ____T

‘Probe He

(c)

.

He ATG

k +

-C--f-

+

-we

Ikb Fig. 1. The pdh gene cloned onto pUC9 plasmid. Small black box indicates

represent HaeIII;

(c) Sequencing

the orientation H, HindIII;

(a) A partial

-

restriction

DNA of 11 kb. (b) A detailed

the position

Phe-16 to Ala-20 in the N-terminal was hybridized.

4

--

I

c

B. sphaerkus chromosomal

-

4-

------

indicates

--

of the synthetic

enzyme map of the plasmid

restriction

oligodeoxyribonucleotide

region of B. sphaericus PDH. The location strategy.

An open arrow

and extent of sequenced

Ne, NaeI; Nr, NarI;

SC, SacI,

regions.

indicates Restriction

and SI, SalI.

pBPDH1.

enzyme map of the SalI-BamHI probe corresponding of the HaeIII

the position

fragment

of the identified

sites: Bm, BarnHI;

BI, Bali;

A blackened fragment

segment

of pBPDH

to the amino acid sequence is indicated,

coding

sequence

1. of

to which the probe of PDH.

C, C/aI; D, DraI;

Arrows

E, EcoRI;

He,

339

-91 CAATGAGCACGATGAAAGCGGTATTCGTTCCATGATGAACGTGACAATGACGTTTGACCATCGTGTCGCCGACGGTGGAACAGTCAATTG CTTTTACAAACCGATTTAAATCClIGAAITGAAGATCCCAAAAA~~~~~~GCTGGAGCTTGTCTGATAAGATTGAATGGAGGAAAAAG~~ 90 70 80 60 40 50 20 30 10 w ATGGCAAAACAGCTTGAAAAGTCATCAAAAATTGGTAATGAGGACGTTTTTCAAAAAATAGCGAATCACGAGCAGATTGTGTTCTGTAAT MAKQCEKSS~~cNr~vfqK~ANHEQlVrCN 180 160 170 150 130 140 120 100 110 GATCCGGTATCCGGCCTGCAAGCTATCATTGCTATCCACGATACAACCCTAGGCCCCGCTTTAGGTGGAACTCGCATGTATCCCTATAAA DPVSGLQAIIAIHDTTLGPALGGTRMYPYK 270 260 240 250 220 230 210 190 200 AATGTGGATGAAGCTCTGGAAGATGTGCTTCGCCTGTCAGAAGGAATGACGTATAAATGCGCAGCCGCCGATATCGATTTCGGCGGCGGG NVDEALEDVLRLSEGMTYKCAAADIDFGGG 360 350 330 340 310 320 300 280 290 AAGGCGGTCATTATCGGAGATCCAGAAAAGGATAAATCTCCGGCATTGTTCCGTGCATTTGGTCAATTTGTGGAATCACTGAATGGACGA KAVIIGDPEKDKSPALFRAFGQFVESLNGR 450 440 420 430 400 410 390 370 380 TTTTACACAGGTACTGACATGGGGACCACGATGGATGATTTTGTCCATGCACAGAAAGAGACGAATTTCATTAACGGAATTCCTGAGCAG FYTGTDMGTTMDDFVHAQKETNFINGIPEQ 540 530 510 520 490 500 480 460 470 TATGGTGGAAGCGGCGACTCGTCGATTCCGACCGCCCAGGGAGTCATTTATGCACTGAAGGCTACAAACCAGTATTTATTTGGAAGCGAT YGGSGDSSIPTAQGVIYALKATNQYLFGSD 630 620 600 610 580 590 570 550 560 AGCCTTTCAGGTAAAACATATGCTATTCAAGGGCTGGGAAAAGTAGGGTATAAAGTAGCGGAACAGCTCTTAAA,~GCCG~CGCCGATTTA SLSGKTYAIQGLGKVGYKVAEQLLKAGADL 720 710 690 700 670 680 660 640 650 TTTGTAACGGATATACATGAAAATGTCCTCAATTCCATTAAGCAAAAATCAGAAGAGCTTGGCGGTTCAGTGACCATTGTAAAAAGTGAC FVYDIHENVLNSIKQKSEELGGSVTIVKSD 810 800 780 790 760 770 750 730 740 GATATTTACAGCGTACAAGCGGATATATTTGTTCCGTGTGCGATGGGTGGTATTATCAATGATAAAACCATTCCTAAGTTAAAGGTGAAG DIYSVQADIFVPCAMGGIINDKTIPKLKVK 890 900 870 880 850 860 840 820 830 GCTGTTGTGGGATCAGCCAATAACCAGCTCAAAGACCTCCGCCATGCAAATGTACTAAACGAAAAGGGAATTCTATATGCACCCGATTAT AVVGSANNQLKDLRHANVLNEKGILYAPDY 980 990 960 970 940 950 930 910 920 ATCGTCAATGCCGGCGGCTTGATCCAGGTTGCTGACGAACTTTATGGGCCGAATAAAGAGCGGGTCTTGCTCAAAACGAAAGAAATTTAC IVNAGGLIQVADELYGPNKERVLLKTKEIY 1080 1050 1060 1070 1030 1040 1020 1000 1010 CGTTCTCTGCTTGAAATTTTTAATCAGGCAGCCCTTGACTGCATCACAACAGTGGAGGCCGCAAATAGGAAGTGTCAAAAGACGATTGAG RSLLEIFNQAALDCITTVEAANRKCQKTIE 1090 1100 1110 1120 1140 1130 1170 GGCCAGCAAACCCGTAATAGTTTCTTTTCTAGGGGACGCAGGCCGAAGTGGAACATAAAAGAGTAATATTGAAAGCGTAAACATTGGAAG G Q Q T R N S F F S R G R R P K W N I K E ++ 1260 AGGAGCTGAACATATGAACAAATATGAAACGATTGATCTTATGGAGGTGGCCAATAATGGGGCCACTCCTCCAAATTGTGATCTTACCTT GCAAATCCAGCCTGTTCATGCAAAGGATGGAAAATCAAAAGGAATTTGGGAGGTAGATGA Fig. 2. Nucleotide letter of the putative Underlined

sequence

of the pdh gene. An ORF of 1143 bp (381 aa) is shown with the deduced

translation

start codon is designated

are those amino acid residues

region, a solid underline pdh promoter.

indicates

as + 1. An arrow and asterisk

which are found in the N-terminal

a potential

ribosome-binding

sequence

site, and the dashes

indicate determined

underline

amino acid sequence.

the start and stop codons,

The first

respectively.

with purified PDH. In the 5’-flanking

the ‘-35’ and ‘-10’ regions of the putative

340

of twenty-four

1Cmers.

This probe was synthesized

according to the amino acid sequence, Phe-16-GlnLys-Ile-Ala-20, which was found in the N-terminal amino

acid sequence

of the purified

PDH (Y.A., R. Matsumoto

and M.O., unpublished

results). Southern-blot fragments of pBPDH1

analysis showed

labeled

hybridized

probe

mixture

Hue111 fragment

indicated

B. sphaericus

of the restriction that the 32P-endto the

0.5-kb

in Fig. lb (not shown).

positive

bacteria,

Denatured

pUC9 plasmid

DNAs carrying restric-

(Graves

Graves and Rabinowitz, 1986). A G + C-rich stem-and-loop

cited

therein).

Rather,

appears to be polycistronic, codon starting at nt position ORF

consisting

of more

than

ACKNOWLEDGEMENTS

(c) Flanking nucleotide sequence of the pdh gene

upstream from the start codon (not shown). The hexanucleotide sequences TTGAAT starting from nt position -67 and ATTAAT from nt position -46 closely resemble the consensus promoter sequences (TTGACA and TATAAT) in the ‘-35’ and ‘-10’ regions for major vegetative promoters of Gram-

100 codons

(not

starting at nt position

We thank Professor Hiroyasu Utiyama of Hiroshima University for thoroughly editing the manuscript. We are also grateful to Ms. Chikako Okubo-Yamada for the syntheses of the primer and probe.

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