Characterization of malate dehydrogenase from deep-sea psychrophilic Vibrio sp. strain no. 5710 and cloning of its gene

ELSEVIER

FEMS Microbiology

Letters I37

( 1996) 247-252

Characterization of malate dehydrogenase from deep-sea psychrophilic Vibrio sp. strain no. 57 10 and cloning of its gene Moriya Ohkuma a’*, Kuniyo Ohtoko “c, Nobuhiko Takada ‘, Tetsuo Hamamoto a3b, Ron Usami ‘, Toshiaki Kudo ‘, Koki Horikoshi bqc ’ Microbiology

Laboratory,

h Deepstar

The Institute

Program,

’ Department

of Physical

Japun Murine

und Chemical

Research (RIKEN),

Science and Technology

of Applied Chemistry.

Toy

Received 6 February

Wake, Saitama 351.01.

Japan

Center, Wake, Saituma 351-01. Japan

Unir~ersit~. Kawgoe.

1996; accepted 6 February

Saitama 350. Japan

1996

Abstract A metabolic key enzyme malate dehydrogenase (MDH) was purified from a deep-sea psychrophilic bacterium, Vibrio sp. strain no. 57 IO. The enzyme displayed an optimal activity shifted toward lower temperature and a pronounced heat lability. A gene encoding this enzyme was isolated and cloned. Recombinant Escherichia coli cells harboring the isolated clone expressed MDH activity with temperature stability identical to that of the parental psychrophile. Nucleotide sequencing of the gene revealed that its primary sequence was similar to that of a mesophile E. coli MDH (78% amino acid identity), for which the three-dimensional structure is known. The enzyme is thus suitable for the analysis of molecular adaptations to low temperatures. Kewvrds;

Psychrophile;

Vibrio;

Deep-sea; Malate dehydrogenase

1. Introduction

Psychrophiles which are able to grow at low temperatures are found in a variety of low-temperature environments, including the deep-sea which is typically a constant low-temperature environment [2,5]. We have obtained a number of psychrophiles and psychrotrophs (psychrotolerants) from various environments and have been studying properties of their extracellular enzymes [4,5]. One of the psychrophiles, designated strain no. 57 10, was identified as a Vihrio species and was found to possess an

s Corresponding author. Tel.: +8l (48) 462 I II I ext. 5724: Fax: + 81 (48) 462 4672; E-mail: [email protected] 0378.1097/96/$12.00 0 1996 Federation PII SO378- 1097(96)00065I

of European

Microbiological

increasing ratio of the polyunsaturated fatty acid, eicosapentaenoic acid (22:6), as an adaptation to maintain membrane fluidity at low growth temperatures [7,8]. Although changes in total fatty acid composition may be a common cold adaptation, physiological adaptations among psychrophiles are considered to be diverse and regulated by a variety of biochemical mechanisms. In particular, coldadapted psychrophiles possess enzymes which are kinetically efficient at ambient environmental temperatures [9,11,12]. The study of the physical structures and catalytic parameters of these cold-active proteins is still incomplete, and the molecular basis of protein adaptation to low temperatures remains unexplored. Societies.

All rights reserved

In order to study the adaptation of cellular metabolism of Vihrio sp. strain no. 5710 to low temperatures, we purified and characterized a key metabolic enzyme, malate dehydrogenase (MDH: Loxidoreductase. EC I. I I .37). for malate:NAD+ which catalytic activities and structures have been well characterized in many organisms. We also cloned and sequenced the gene encoding MDH in order to compare the primary structure of the enzyme with those of other organisms and to elucidate possible molecular changes important in cold adaptation.

2. Materials

MDH activity was measured spectrophotometritally by the increase in absorbance of NADH at 340 nm after the addition of an appropriate amount of enzyme to an assay mixture containing 100 mM glycine-NaOH buffer (pH 10.0). I .23 mM NAD’. and 100 mM sodium malate in a final reaction volume of I ml adjusted with the addition of distilled water. To observe oxalacetate reduction, the activity of a reaction mixture containing 40 mM potassium phosphate (pH 7.0). 0. IS mM NADH, and 0.2 mM oxalacetate was followed by decrease in absorbance of NADH. Protein content was measured with a BCA protein assay kit (Pierce). The thermal stability of the enzymatic activity was measured by incubating the enzyme solution (200 pg/ml) in 40 mM potassium phosphate (pH 7.0) at various temperatures followed by assaying at 30°C with the substrate. Amino acid composition of the purified enzyme was determined after hydrolysis in vacua with 4 N methanesulfonic acid for 20 h at 104”C, and the hydrolyzed sample was analyzed on a Shimadzu LC-9A amino acid analyzer. Peptides obtained by a Residue-specific protease kit (TAKARA) were separated by SDS-PAGE and blotted onto PVDF membrane (BioRad), and the partial amino acid sequences were determined by using an automated Edman

and methods

A deep-sea psychrophilic bacterium Vihr-io hp. strain no. 5710 [6] was used in this study and grown in LB supplemented with 3% NaCl on a gyrorotator} shaker for 24 h at 5°C. At log-phase. the cultures were centrifuged and the resultant cell pellets were stored frozen at - 80°C. MDH was purified from the cells by several steps of chromatography as described in Table 1.

Table

I

Purification of malate dehydrogenahc from Vihio

Crude

\p. atI-ain no. 57 IO

Total protein

Total activity

Specific activity

Recocer)

Purification

(mg)

(kt-1)

(hLl/mg

(9)

(fold)

2000

I 600

I75

IS00

DEALToyopearl redA Superdex 200

protein)

0.8

IO0

I7

93

I II

11.9

I IO0

7.4

67

92

2.7

260

Yh

I6

IX

All purification protocols, were performed at 4°C and all hufferh used in the early hIage\ of purification. except for the final gel filtration step, contained 1 mM PMSF and I mM EDTA. mM Tris

Sixty grams of froren paste of the cells was thawed by adding 300 ml of Tris-buffer

(SO

HCI. pH 8.0). The slurry was subjected to sonic disruption (Tomy Seiko) at the setting of 1 for 30 s, repeated 5 times. The

suspension wa\ centrifuged at 16000 X ,y and the supernatant was saved. The crude supernatant (330 ml) was applied to a DEAE-Toyopearl column (Toyo Soda; 20 by 2.2 cm, 300 ml), previously equilibrated with the Tris-buffer. buffer, enzyme was eluted with 2

1(linear

After washing the column with 600 ml of the bamc

gradient) of 0 to 500 mM NaCI in the Tris-buffer. The enzyme wus eluted from the column at 200

to 230 mM NaCI. The fraction\ containing the enzyme activity were pooled and directly applied to a redA affinity column (Amicon:

IO by

2.2 cm) previously equilibrated with Tris-buffer. The column wab uashed with Tris-buffer until materials with A?,,, absorbance were eluted from the column. The enzyme activity was then eluted with Tria-huffer concentrated with Centriprep

IO and Centricon

Superdex 200 column (Pharmacia). same buffer in a Pharmacia

FPLC

IO (Amicon)

containing I mM NADH.

previously equilibrated with Tris-buffer \y\tem

and the active

The active fractions were pooled and

to a final volume of 300 ~1. The concentrated hampIe wa\ injected into a

fractions

were pooled

containing

100 mM N&l.

as a purified

enzyme

The enzyme was eluted with the

preparation.

M. Ohkuma et al. / FEMS Microbicdog?

degradation system of an ABI protein model 47314 (Applied Biosystems). 2.2. PCR ampl$cation, quencing

sequencer

cloning and nucleotide

se-

The partial MDH gene was amplified from the chromosomal DNA of strain 5710 by PCR using Taq DNA polymerase (TAKARA) according to manufacturer’s directions, except that 20 mM MgCl, was used. The PCR primers used were 99F (5’TAYGAYATHGCNCCNGT-3’) and 730R (5’ACDATNCCYTTYTCNCCYTG-3’), where Y represents C or T, H represents A or C or T, N represents A, G, C or T, and D represents A, G or T. Degenerate primer sequences were derived from amino acid sequences of YDIAPV for 99F and of QGEKGIV for 730R. The PCR reaction profile was for 25 cycles at 94°C for 20 s, 55°C for 20 s and 72°C for 2 min. Chromosomal DNA was digested with several restriction enzymes, electrophoresed by 1% agarose gel electrophoresis, blotted onto a Hybond-N+ membrane (Amersham) by vacuum blotting and analyzed by Southern-blot hybridization using the PCRamplified product as a probe. Detection of hybridized DNA bands was performed with a DIG DNA detection kit (Boehringer). A 2.4 kb HaeIII

1 2

,::!a,

21 K 14 K,:

T, ‘!b

Fig. 1. SDS-PAGE of the purified MDH. The purified applied to a 7%15% gradient polyacrylamide-SDS trophoresia and the polypeptide band was identified by Blue staining. Lane 1, molecular mass markers; lane MDH. Molecular masses of markers are described on

247-282

MDH was gel elecCoomassie 2, purified the left.

249

fragment which hybridized to the PCR product was separated by agarose gel electrophoresis, purified from the gel, ligated into the SmaI site of pUC 119 and transformed into Escherichia coli. The transformants were screened by colony hybridization using the PCR product as a probe with the DIG DNA detection kit. Deletion derivatives of the isolated clone were obtained by a Kilo-sequencing deletion kit (TAKARA) and both strands with appropriate overlaps were sequenced with an ABI 373A DNA sequencer using a Tag dye primer cycle sequencing kit (Applied Biosystemsl. The nucleotide sequence data will appear in the DDBJ, EMBL, GenBank nucleotide sequence databases under the accession number D78 194.

3. Results and discussion 3.1. Pur$cation

qf MDH

The MDH was purified to homogeneity from the cells of Vibrio sp. strain no. 5710 (strain 5710), and the results of the purification are summarized in Table 1. The overall recovery was 16% with a purification factor of 120. The purity of the enzyme was confirmed by SDS-PAGE (Fig. 1) and gel filtration chromatography where the purified enzyme yielded a single band and a single peak, respectively. The molecular masses of the MDH deduced by SDS-PAGE and gel filtration chromatography were 35 000 and 72000, respectively, indicating that the native enzyme may be composed of two identical subunits. 3.2. Characteristics

43 K 30K

Letters 137 (I9961

of the purified MDH

Several catalytic properties of the purified MDH from strain 5710 were determined (summarized in Table 2). The enzyme displays an apparent optimal activity shifted toward low temperature and a pronounced heat lability. The enzyme had an optimal temperature for activity of 35°C which was approximately 15°C lower than that of E. coli MDH determined by using the crude extract. At 10 and 2O”C, the enzyme maintained 27 and 40% of the activity at the optimal temperature, respectively. No activity

250

M. Ohkurntr rt trl. / FEMS Micmhiolog~

Lrttrc

was observed after 20 min at 45°C due to thermal inactivation, while the MDH activity in the E. coli extract retains near total activity after 30 min exposure at 50°C. Similar optimal activity at low temperatures and heat lability have been observed with other psychrotrophic enzymes [4,9, I 1,121. Despite the psychrotrophic temperature activity profile, other catalytic properties are similar to MDHs from mesophiles. Thus, the MDH from strain 57 10 showed the characteristics of a typical psychrotrophic enzyme. Partial amino acid sequences in the N-terminus and three other parts of the MDH were determined and the amino acid composition was also obtained. 3.3. Molecular

properties

pPM125 5.6 kbp

MDH

\

BamHl

cloning of MDH gene

Based on partial amino acid sequences, several degenerate PCR primers were designed and used to amplify MDH gene from the chromosomal DNA of strain 5710. Among tested combinations of the PCR primers, a combination of the primers, 99F and 730R, produced a single amplified product of 650 bp in length. Using the 650 bp PCR product as a probe. Southern-blot hybridization of the chromosomal DNA gave single bands in several restriction digestions. A 2.4 kb Hue111 fragment which hybridized with the PCR product by genomic Southern-blot

Table 2 Catalytic

I37 ClYY61 247-252

of MDH from Vibrio

Temperature optimum pH optima malate dehydrogenation oxalacetate reduction Isoelectronic point K, values at 30°C L-malate oxalacetate NAD + NADH

sp, strain no. 57 IO

35°C pH 9.5 pH 8.0 p1 6.3 1.5X lo-’ M 0.5X lo-’ M 1.5~ IOmF M 1.0X IOmi M

Enzyme optimal temperature was determined by incubating the reaction mixtures at various temperatures in 40 mM potassium phosphate (pH 7.0). To determine enzyme optimal pH, the enzyme solution was incubated at 30°C in 40 mM reaction mixtures at various pHs (acetate buffer between pH 5.0 and 6.0, phosphate buffer between pH 6.0 and 7.5, Tris. HCI buffer between 7.5 and 9.0, and glycine-NaOH buffer between pH 9.0 and 1 I .O). Isoelectronic focusing was performed with a PhastSystem (Pharmacia) as described by the supplier.

Fig. 2. The restrictlon map of pPM125. Black box and big arrow indicate the 2.3 kbp HwIII fragment cloned in pPM125. The MDH coding region is indicated by big arrow. Thin line. thin box (ori) and thin arrow (Ampr) correspond to the sequence of pUCl19.

analysis was cloned. Fig. 2 shows the restriction map of the isolated clone pPM125. The E. coli strain harboring pPM125 produced &fold more MDH activity than an E. coli strain harboring control plasmid pUC 119. After thermal inactivation for 30 min at 40°C. the MDH activity of the E. coli strain harboring pPM 125 was decreased to approximately the same level as that of the control E. coli strain. MDH in the E. co/i strain harboring pPM125 possessed the same temperature stability profile as that of the parental psychrophilic strain 5710, indicating that the MDH gene of strain 57 IO was cloned and expressed in E. cd. 3.4. Nucleotide

sequencing

of MDH gene

The nucleotide sequence of the cloned fragment in pPM 125 was determined. The sequence was found to contain an open reading frame encoding 3 12 amino acids in the central part of the cloned fragment. It was confirmed that the reading frame corresponded to that of MDH as described below. The molecular mass of the protein encoded by this coding sequence was calculated to be 32000, which was similar to that of the purified enzyme subunit (Mr 35 000) measured by SDS-PAGE. The identical sequences with partial amino acid sequences of the

M. Ohkuma et al. / FEMS Microbiology

5710 Pb EC St Pm

MKVAVLGAA:GIG OALAL LFKT LP I

NG A

PS

s

A

Pl 5710 Pb Kc St Pm

AKK AKK PA-

A 9

!z

N

s ii

NS-

L”

R

H-

A

E

ac

v

R

v

”

K C LPP

CCC'

I33

T

I32

cm

SEAP-E SEAP-E YL PEQLPDC

A

TD

T

100p

84

”

TVA

ClD/E

5710 Pb Ec St Pm 160 170 180 ~~FVSALKGISLADVEVPVIGGHSGVTILPLLSQ-VEE I AE XTPSELQ

150

5710 Pb EC St Pm

I N I NP I" ANA

AE AE AE

XQPGE KLPTE LDP R s ~

a2F 200

AK

190

I

P7 210

I

-E -P -IP CTPX

SD s EQ S EQ D PQDQ

f+3

110

110

1.0

5710 Pb Ec St Pm

WALTARIQNAGT~EAKAGGGSATLSMGQ-FGLSLVREKGI IX P R Y ADK AAE X LST G E X A AY G

5710 Pb EC St Pm

VECTYVDGGSEHATFFAQPVLLGXNG"EE"L&YGELSEFETNARDA"LEE AEDGXR D ID X T QELNN DT A EDGQYR s L RKSI T A QN LEG DT A EDGQYR s L RXSI T A QHSL DT SF XSQETDCPY ST L X II(NI XI P EXMIAEAIP

5710 Pb Ec St pm

09 300 310 LXANITLGEEFVA--G A --X TSD XDA Q N--X IIN--K XD Q S XX KNMK

alG/2G 150

QQ QV V Vk

" DMNXE;

a3G 260

270

PlO

280

p11

290

-~-

cm

Fig. 3. An alignment of five MDH amino acid sequences. MDHs from Vibrio sp. strain no. 5710 (57101, Photobacterium sp. strain SS9 (Pb), E. coli (EC), S. ~phimurium (St), and porcine heart mitochondria (pm) are aligned. Only amino acids different from those of 5710 MDH are shown in Pb, EC, St, and pm. The predicted secondary structures based on the three-dimensional analysis of E. coli and porcine heart mitochondrial MDHs are shown under the alignment. Identical amino acids with the peptide sequencing of the purified enzyme are underlined in the 5710 MDH sequence.

purified enzyme were found in the amino acid sequence deduced from the DNA sequencing (Fig. 3, underlined). The amino acid composition of the MDH calculated from the nucleotide sequence was in good agreement with that obtained by chemical analysis of the purified enzyme (not shown). 3.5. Comparison

of amino acid sequence of MDH

The amino acid sequence of the MDH from strain 5710 (5710 MDH) was compared with those of

Letters 137 (19961247-252

251

known three-dimensional structure. Among them, MDHs of E. coli [3] and porcine heart mitochondria [l] showed relatively high amino acid identities, 78.1% and 56.3%, respectively, with 5710 MDH. In the databases, MDHs from a deep-sea psychrophilic Photobacterium sp. strain SS9 (GenBank accession number, L133 19) and from Salmonella typhimurium [lo] showed high amino acid identities, 78.8% and 76.5%, respectively. An alignment of amino acid sequences among MDHs is shown in Fig. 3. The similarities were not evenly spread over the entire sequences, but were clustered within regions. The alignment of the amino acid sequence of 5710 MDH with those of MDHs of known three-dimensional structure [ 1,3] allows the identification of functionally important residues and domains. The His- 177 and Asp- 150 represent the signature residues forming the His-Asp pair in the active site of MDH. The Arg-81, Arg-87, Arg-153, and Asn-119 are thought to be situated in the active site and to bind to the substrate by hydrogen bonds. The NH,-terminal region containing the cofactor binding domain (corresponding to six parallel P-sheets) and the regions corresponding to the (YB, aC, cr2F, (w2G, and a3G, which are important in subunit interactions, are all well conserved in 5710 MDH. Although the regions corresponding to p 3, (YC’, CY E, the region between a2F and /3 7, (YlG, and the C-terminal region are relatively variable, these regions correspond to surface regions of the protein molecule, which may be less constrained than active sites or regions involved in subunit interaction. The simplest view of the enzyme adaptation to catalysis at low temperatures is to postulate that the psychrotrophic enzyme possesses a more flexible structure in order to undergo rapid and reversible conformational changes in low temperatures as required by the catalytic cycle [9]. Thermal instability is regarded as the consequence of the highly flexible structure of cold-active enzymes. Considering that the amino acid sequence of 5710 MDH is closely related to that of the E. coli MDH enzyme of which three-dimensional structure has been determined, the psychrotrophic Vibrio MDH is ideally suited for the analysis of molecular adaptations required to compensate for the reduction of enzyme activity inherent to low temperatures.

252

M. Ohkumo rr ul. / FEMS Microbiology

Acknowledgements The authors thank Drs. M. Roberts and M. Travisano for critical reading of the manuscript. This work is partially supported by grants for the Biodesign Research Program and the Genome Research Program from RIKEN to M.O. and T.K.

References [I] Gleason, W.B.. Fu, Z., Birktoft, J. and Banaszak, L.J. (1994) Refined crystal structure of mitochondrial malate dehydrogenase from porcine heart and the consensus structure for dicarboxylic acid oxidoreductases. Biochemistry 33, 2078 2088. [2] Gounot, A.-M. (1991) Bacterial life at low temperature: physiological aspects and biotechnological implications. J. Appl. Bacterial. 71, 386-397. [3] Hall, M.D., Levitt, D.G. and Banaszak, L.J. (1992) Crystal structure of Escherichia coli malate dehydrogenase. J. Mol. Biol. 226, 867-882. [4] Hamamoto, T. and Horikoshi, K. (1991) Characterization of an amylase from a psychrotrophic Vibrio isolated from a deep-sea mud sample. FEMS Microbial. Lett. 84. 79-84.

Letters 137 (19961 247-252

[5] Hamamoto. T. and Horikoshi, K. (1993) Deep-sea microbiology research within the Deepstar program. J. Mar. Biotechnol. I. 119-124. [6] Hamamoto, T., Takata. N., Kudo, T. and Horikoshi, K. (I 994) Effect of temperature and growth phase on fatty acid composition of the psychrophilic Vibrio sp. strain no. 5710. FEMS Microbial. Lett. 119. 77-82. [7] Hamamoto, T., Takata. N., Kudo, T. and Horikoshi, K. (1995) Characteristic presence of polyunsaturated fatty acids in marine psychrophilic vibrios. FEMS Microbial. Lett. 129. 5 I-56. [8] Hochachka. P.W. and Somero, G.N. (1984) Biochemical Adaptations, Princeton University Press, Princeton, NJ. [9] Langridge, P. and Morita, R.Y. (1966) Thermolability of malic dehydrogenase from the obligate psychrophile Vibrio marinus. J. Bacterial. 92, 418-423. [IO] Lu, C-D. and Abdelal, A.H.T. (1993) Complete nucleotide sequence of the Salmonellu r~phimurium gene encoding malate dehydrogenase. Gene 123, 143-144. [I I] Mitchell, P., Yen, H.C. and Mathemeier, P.F. (1985) Properties of lactate dehydrogenase in a psychrophilic marine bacterium. Appl. Environ. Microbial. 49. 1332-1334. [12] Ochiai, T., Fukunaga, N. and Sasaki, S. (1979) Purification and some properties of two NADP+-specific isocitrate dehydrogenases from an obligately psychrophilic marine bacterium, Vibrio sp., strain ABE-l. J. Biochem. 86, 377-384.