THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 49, Issue of December 9, pp. 30888-30892, 1994 Printed in U.S.A.

Fatty Acyl Transfer by Human N-Myristyl Transferase Is Dependent Upon Conserved Cysteine and Histidine Residues*

(Received for publication, August 29, 1994, and in revised form, October 4, 1994)

Steven M. PeseckisS and Marilyn D. Reshs From the Cell Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

N-Myristyl transferase (Nrnt) catalyzes attachment of myristate onto the N terminus of suitable proteins. In order to identify amino acids important for catalytic functions, human Nmt and mutants representing all six conserved cysteine and histidine residues (Cys-169, Cys- 214, His-131, His-171, His-218, and His-293) were ex- pressed in Escherichia coli and analyzed for their ability to bind and transfer myristic acid. N-Terminal histidine- tagged fusion proteins displayed varying abilities to form an association with radiolabeled myristic acid in- dicative of an acyl-enzyme intermediate. When co-ex- pressed with an acceptor substrate protein, pp6OV"", the mutants showed differential incorporation of radiola- beled myristic acid into v-Src protein. In vitro experi- ments monitoring transfer of myristyl CoA to a peptide homologous to the N terminus of pp60""" gave results similar to those obtained in uiuo. Our studies showed that mutation at Cys-169, His-171, and especially His-293 interfered with formation of an acyl-enzyme intermedi- ate, while human Nmts containing mutations at Cys-169, His-218, or His-293 showed greatly attenuated abilities to form acylated product. We propose a model for the Nmt reaction mechanism in which Cys-169 serves as the fatty acid attachment site for a covalent myristyl en- zyme intermediate, while His-171 acts as a general acid base and His-293 as a specific acidhase during acyl-en- zyme intermediate formation. His-218 could then act as an acid or base needed to catalyze transfer of the acyl group from the acyl-enzyme intermediate to a polypep- tide substrate. This working model will be useful for the design of regulators of Nmt function.

N-Myristyl transferase (Nrnt)' is a unique eukaryotic enzyme responsible for catalyzing the formation of an amide bond be- tween myristic acid and the N-terminal glycine of select polypeptides (1). Studies of yeast and human enzymes indicate that Nmt is encoded by a single copy gene ( 2 4 ) a n d that i t functions in a cotranslational manner to myristylate its protein

* This work was supported by National Institutes of Health Grant CA-52405. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertzsement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

lege of Pharmacy, University of Toledo, Toledo, OH 43606-3390. f Present address: Dept. of Medicinal and Biological Chemistry, Col-

5 Rita Allen Foundation scholar and an Established Scientist of the American Heart Association. To whom correspondence should be ad- dressed: Cell Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., Box 143, New York, NY 10021. Tel.: 212-639-2514; Fax: 212-717-3317; E-mail: m-resh@ski.mskcc.org.

The abbreviations used are: Nmt, myristoyl-CoAprotein N-myris- toyltransferase; hHmt, human myristoyl-CoAprotein N-myristoyl-

transferase; PCR, polymerase chain reaction; IPTG, isopropyl-1-thio+- transferase; Nmtlp, S. cerevisiae myristoyl-CoA:protein N-myristoyl-

dz-galactopyranoside; SDS, sodium dodecyl sulfate; PAGE, polyacryl- amide gel electrophoresis; AMP, ampicillin.

substrates (5,6). The incorporation of myristate into the N ter- minus of a number of oncogene, viral, and eukaryotic gene pro- tein products is indispensable for their protein function (1, 7).

Human Nmt (hNmt) exhibits a sequential ordered bi-bi re- action mechanism (8,9). For Saccharomyces cerevisiae N-myris- tyl transferase (Nmtlp), an intermediate of fatty acyl coenzyme A (CoA) associated with the enzyme can be detected by isoelec- tric focusing (change in PI from 8.15 for apoenzyme to 6.7 for bound form) or fluorography of SDS-PAGE gels (binding of radiolabeled myristyl-CoA) (10). Chymotryptic digestion of the fatty acyl-CoANmtlp complex produces fragments that retain labeled myristic acid but that are hydrolyzable by hydroxyla- mine (10). These results suggest that, in S. cerevisiae Nmtlp, a covalent intermediate forms that involves a serine or cysteine residue located between Asn-42 and Thr-220 (hNmt region Asp-41 to Trp-217) (11). Comparisons among four yeast and one human Nmt protein sequences reveal that 2 cysteine residues, at amino acids 169 and 214 in hNmt (172 and 217 in Nmtlp; see Fig. 11, are conserved (2-4, 12). While early chemical char- acterization of the yeast transferase seemed to preclude the involvement of these cysteines in the transfer mechanism (131, more recent results with hNmt using iodoacetamide and N-5,5'-dithiobis(2-nitrobenzoic acid) implicate some cysteine requirement (14). In addition, 4 histidine residues are con- served (see Fig. 1; His-131, His-171, His-217, and His-293 in hNmt; His-134, His-174, His-221, and His-296 in Nmtlp). As noted by Towler et al. (13) and McIlhinney et al. (141, one or more of these histidines could be involved in the transfer mech- anism since the rate of in vitro transfer of the acyl group to glycine significantly increases between pH 6.5 and 7.5 and the reaction can be fully inhibited by diethyl pyrocarbonate.

In this study, we have identified and mutated the conserved cysteine and histidine residues in hNmt and expressed the mutants in Escherichia coli, an organism that has neither en- dogenous Nmt nor suitable substrates (6,15,16). These results allowed us to delineate specific contributions of the conserved amino acids to the enzyme transfer mechanism.

EXPERIMENTAL PROCEDURES Materials-The plasmids pETllc and pET19b and BL21 DE3 bacte-

ria were purchased from Novagen. Human hippocampus cDNA library in A Zap I1 vector and pBluescript KS- plasmid were from Stratagene. Taq polymerase (Perkin-Elmer Corp.) and Deep Vent DNA polymerase (New England Biolabs) were used for PCR. [3HlMyristic acid, sodium [35S]sulfate, [Y-~~PIATP, and [35Slmethionine were obtained from DuPont NEN. The hNmt cDNA employed encoded a polypeptide se- quence identical to that reported (3) but was obtained as described below.

Identification of hNmt cDNA Sequence in Human Hippocampus Library by PCR-cDNAfragments suitable for use as probes in screening of a cDNA library for genes encoding hNmt or possible isozymes were obtained by PCR. Ahuman hippocampus cDNAlibrary in A ZAP I1 (Strat- agene) was employed as template for PCR using degenerate primers based on conserved regions of Nmt cDNA sequences (see Fig. 1) (24,121. The criteria for isolating cDNA related to Nmt was the simultaneous presence ofthe conserved sequences NYVED (hNmt amino acids 99-103, VEINF (hNmt amino acids 163-167), and PVLI(R/K) (hNmt amino acids

30888

Human N-Myristyl Transferase Active Site 30889

181-185). Five oligonucleotide primers were synthesized. Primer 1 con- sisted of a spacer, EcoRI site, and degenerate codon usage for the coding cDNAfor NYVED: 5'-CGGAATTCAA(TC)TA(TC)GT(ATCG)GA(AG)GA- 3'. Primer 2 consisted of degenerate codon usage for the noncoding cDNA for VEINF, a HindIII site, and spacer: 3'-CA(ATCG)CT(TC)TA(AGT)T- T(AG)AA(AG)T"CGAACG-5'. The third primer consisted of a spacer, EcoRI site, and degenerate codon usage for the coding cDNA for VEINF (hNmt amino acids 163-167): 5'-CGGAAT"CGT(ATCG)GA(AG)AT(AT- C)AA(TC)TT-3'. Finally, for the last sequence, two primers were required due to the six possible coding combinations for leucine. Thus, the fourth and fiRh primers were synthesized consisting of degenerate codon usage for the noncoding cDNA for PVLKFUK), a HindIII site, and spacer: 3'- GG(ATCG)CA(ATCG)GA(ATCG)TA(ATG)[GTXCT)TTCGAACG-5' and

products were cut with EcoRI and HindIII and ligated into pBluescript KS-. Cloned DNA in DH5a bacteria was isolated and sequenced. The clones obtained by PCR resulted in a cDNAfragment encoding a polypep- tide sequence identical to that of liver hNmt between amino acids 104 and 162 (3).

Screening ofHuman Hippocampus Library with Unique Oligonucleo- tide Probes-Two nondegenerate oligonucleotides suitable as probes for library screening were synthesized. Probe 1, 20 oligonucleotides (GAA AAC ACC CGAGAG GCC GG, complementary to the sequence encoding hNmt amino acids 117-123: LLWALRP), and probe 2, 30 oligonucleo- tides (GTG ACA CCC CAA GCT CAC CAG AGT TCA GCC, complemen- tary to the sequence encoding hNmt amino acids 131-140: HCGVRV- VSSR), were labeled with 32P by incubation for 40 min at 37 "C in the presence of [y-32P]ATP and T4 polynucleotide kinase followed by puri- fication through Chromospin-10 columns. These radiolabeled probes were used in the library screening.

Sixteen 150-mm plates each with 40,000 plaques of a human hip- pocampus cDNA library in A ZAP I1 (Stratagene) produced upon infec- tion ofXL-1 Blue bacteria were grown for 8 h a t 37 "C. Two replicates of each plate were produced by blotting onto Biotrans Nylon Membranes (ICN) followed by alkaline denaturation, neutralization, and immobili- zation by UV cross-linking (Stratagene Stratalinker UV).

One set of replicates was incubated with probe 1, the other with probe 2. Autoradiographs of each washed replicate set were compared for clones to which both probes hybridized. Incubations and washes were as described in Berent et al. (17). Comparison of autoradiographs of replicates indicated 12 possible clones. Phages containing each pos- sible clone were eluted from the plate agar into SM buffer (100 mM NaC1,S mM MgSO,, 50 m~ Tris-HC1, pH 7.5, 1% gelatin). Two dilutions of each candidate phage were used to infect XL1-Blue bacteria, which were grown in top agar to produce 30-150 plaques/plate. Two replicates of each plate were prepared and probed as described (17). Only one clone was strongly labeled by both probes.

The desired cDNA was excised from the A Z A P I1 and isolated as an insert in pBluescript SK- phagemid vector following Stratagene's rec- ommended procedure. Sequencing of the isolated clone indicated that i t encoded a protein identical to that reported for hNmt isolated from a human liver cDNAlibrary (3) except for a deletion of 77 base pairs in the terminal 5' cDNA coding region and the artifactual inclusion of an additional gene following the 3' region. We used nondegenerate oligo- nucleotides based on the reported 5' coding cDNA sequence and several 3' noncoding cDNA sequences of hNmt as primers for PCR. When the entire human hippocampus cDNA library was used as template, no full-length clones were detected in the library. Consequently, the full- length gene was obtained by insertion of a synthesized oligonucleotide encoding the published 5' hNmt region (3) (which was not present in the Stratagene library) and removal of the extraneous 3' gene.

Construction of a Plasmid for Protein Co-expression in E. coli-A single pET11-based plasmid co-expressing hNmt and pp60""" was con- structed. The v-src gene was derived from a pET19 plasmid in which wild type v-src was inserted 5' into the NcoI and 3' into a blunted NdeI site of pET19b. The BamHI site of pET19 with v-src was removed by digestion with BamHI, made blunt with Klenow, and religated with T4 DNA ligase. Using the sense 5'-AGATCTCGATCCCGCGAAAn'AAT- ACG-3' and antisense 5'-GATATCCGGATATAGTTCCTCC-3' primers and Deep Vent polymerase, the region encoding the T7 promoter, v-SI% gene, and T7 terminator regions were amplified by PCR. Plasmid pETllc was cut with BglII and blunted with Klenow. The PCR product from PET19 with v-src was ligated with T4 ligase into the pETllc- blunted BglII site. The gene for hNmt, wild type and mutants, was inserted between the NdeI and BamHI sites. The final plasmid thus contains v-src 5' to Nmt, each with distinct T7 promotor and termina- tion regions, and oriented for transcription in the same direction.

3'-GG(ATCG)CA(ATCG)AA(TC)TA(ATG)(GT)(CT)~CGAACG-5'. PCR

Mutagenesis-Nmt mutants were generated by PCR using Deep Vent polymerase (18). A sense oligonucleotide corresponding to the hNmt N-terminal region and an antisense mutant oligonucleotide, and sepa- rately an antisense oligonucleotide corresponding to the hNmt C ter- minus with a sense mutant oligonucleotide were amplified by PCR. Purified PCR product from each amplification was combined with hNmt N- and C-terminal sense and antisense oligonucleotides, respectively, and amplified by PCR to give full-length Nmt genes. Genes cut with HindIII and HincII were inserted into pBluescript KS- for clone selec- tion and sequencing and then with NdeI and BamHI for insertion into pETllc, pET19b, and pETll+v-src. Mutant H171N was cloned directly into pETllc. The cDNAs of all mutants were sequenced from end-to-end after PCR, the only mutations observed were those intentionally intro- duced to create each desired mutation.

Protein Induction-PET vectors containing inserts were used to transform BL21 DE3 bacteria. Transformed bacteria were grown over- night in LB media with 200 pg/ml ampicillin (LBIAMP). Eighty pl of each overnight culture in an Eppendorf tube was spun at 14,000 x g for 4 min. The supernatant was discarded, and the pellet was resuspended in 100 pl of mediaJAMP (LB or M9/AMP) and respun. The pellet was resuspended in 1 ml of medidAMP in a 17 x 100-mm glass culture tube and incubated at 37 "C and 260 rpm for 1 h. The tube was spun 10 min at 2000 x g. The pellet was resuspended in 1 ml of mediaJAMP with 1 mM IPTG. The tube was incubated at 37 "C and 260 rpm for 2 h, the A,,, measured, and then spun for 10 min at 2000 x g to form a bacterial pellet.

Protein Labeling-30 pl (30 pCi) of [3H]myristic acid in ethanol was added to a glass culture tube and reduced to a residue by a nitrogen stream. Bacteria in media with IPTG were added to the tube. After induction, the bacterial pellet was lysed with 1 x sample buffer con- taining 10 mM dithiothreitol and analyzed by SDS-PAGE. For 35S label- ing, sodium [35S]sulfate in water was added to M9/AMP with IPTG.

In Vitro Assay-The bacterial pellet was resuspended in 100 pl of TDE buffer (10 mM Tris, pH 7.4, 1 mM dithiothreitol, 0.1 mM EDTA, 10 pg/ml aprotinin, 0.3% Triton X-100) with 1 mg/ml lysozyme and incu- bated a t 23 "C for 1 h. Bacteria were sonicated until nonviscous and spun at 14,000 x g for 4 min. Approximately 10 p1 of supernatant was mixed with 40 pl of [3Hlmyristyl-CoA, 5 p1 of 5 mg/ml GYSrc peptide (sequence: GSSKSKPKDPSY) (6), 40 pl of TDE, and 30 p1 of dH,O and incubated a t 37 "C for 30 min. Protein was precipitated with 120 pl of MeOH and 5 pl of trichloroacetic acid, chilled 8 min on ice, and spun at 8000 x g for 4 min, and the supernatant was filtered through a Spin-X filter. The filtrate was analyzed for [3HlMyrGYSrc formation by HPLC over a C,, column using watedacetonitrile with 0.1% trifluoroacetic acid as eluants.

RESULTS AND DISCUSSION

Only One Gene Coding for Human N-Myristyl Transferase Is Observed-In order to isolate the gene for human Nmt, we screened a human hippocampus cDNA library with oligonucleo- tides, as described under "Experimental Procedures." Only one clone of hNmt was isolated, which encodes a protein identical from amino acids 27 t o 416 to that of liver hNmt. No full-length hNmt clones were detected in the library. We therefore gener- ated a complete hNmt cDNA by insertion of a synthetic oligo- nucleotide encoding amino acids 1-26 of liver hNmt (3).

The human hippocampus cDNA library was further screened for potential hNmt isozymes using degenerate primers encom- passing residues 104-162. No other clones were found. These results are in agreement with reported Southern blot analysis (3). Our findings strongly suggest that factors other than the existence of Nmt isozymes account for the incorporation of non- myristic fatty acids into the N terminus of some proteins (19).

The amino acid sequence of hNmt was compared with Nmt sequences from other organisms ( 2 4 , 12). 2 cysteines and 4 histidine residues were found to be conserved among all known Nmts (Fig. 1). These 6 amino acids were mutated (Cys to Ser, His to Asn) in order to evaluate their importance to enzyme activity. Wild type and mutant enzymes were expressed in bac- teria with and without protein substrate and as N-terminal histidine-tagged fusion proteins. Their abilities to form a com- plex with myristic acid in vivo and to myristylate polypeptide substrates in vivo and in vitro were evaluated.

30890 Human N-Myristyl Dunsferase Active Site >>>K.------FW-TQPV---------..-G~----~..------~--L---

F ~ W - - - D - - - - - - - - - - - - L L - - ~ ~ V E D - ~ - - F R F - ~ - - ~ F - - ~ ~ ~ L - - ~ G ~ - -

- I . . ~ G VR-----KLV-~I---~-----..--------EINFL~-~-~~-~~-

- P V L I - E ~ T R R ~ - - - - I - - A - ~ T - ~ - - L ~ - ~ - - ~ ~ ~ ~ - - - - - K L - - " - F -

-L~---T---------~~---K--GLR-----D---~--L---~---F-----

- - - E E - - ~ - - - - - * ^ ^ ^ ^ * - - - - - - W E - - - G - - ~ ~ - - ~ - ~ - ~ ~ - ~ - - -

----"L-"y"y""*^^^^^^*~^*~^*^**"-L"~"DA""

131 169 l i l

214 218

2'13

K----DVFN-L----N--FL---KFG-GDG-GDG-L--~L-N---------**^

A ^ ^ A * ^ ^ ^ A * A " c - v "

0 li,or I I,ctid,m% ~ l \ r , , C \ < a i n c s

FK:. 1. Amino acids conserved among N-myristyl transferase translated cDNA sequences in Homo sapiens, S. cereuisiae, C. albicans, C. neoformans, and H . capsulafum. Residues are num- bered based on the human Nmt sequence. Amino acids conserved in all five Nmt sequences are denoted by capitol letters; those conserved in four out of five Nmts are denoted in lotuercase letters; and . denote where amino acid insertions or deletions, respectively, are observed; >>> indicates variable N-terminal length.

Human N-Myristyl Transferase with an N-Terminal Histi- dine Tag Forms an Acyl-Enzyme Complex-We first generated a fusion protein of hNmt containing 10 N-terminal histidine residues (Hislo-Nmt). Incubation of bacteria expressing HislO- Nmt with ['HH]myristate resulted in the formation of an acyl- enzyme complex in vivo, which was stable to SDS-PAGE. In contrast, no readily identifiable complex was evident when bac- teria expressing unmodified wild type hNmt were similarly incubated with I"H1myristic acid. One explanation for the ob- served result is that the presence of an N-terminal extension may serve to stabilize the acyl-enzyme intermediate. Alterna- tively, specific amino acid residues in the extension could con- tribute stabilizing charge or lipophilic interactions. We ob- served that, under identical conditions, HislO-Nmt was more active in vitro than unmodified hNmt.

The ability to detect a n acyl-enzyme intermediate with HislO-Nmt allowed us to test the effect of mutating conserved cysteine and histidine residues. Mutation of Cys-169, His-171, or His-293 of HislO-Nmt either greatly reduced or prevented formation of a n acyl-enzyme intermediate (Fig. 2), implying that these residues are important for binding of myristate to Nmt. Acyl-enzyme complex formation was relatively unim- pared in the samples containing C214S, H131N, and to a lesser extent, His-218 Nmts.

Only One of 2 Conserved Cysteines a n d 3 of 4 Conserved Histidines Are Involved in the Transfer Mechanism-In order to identify residues important for transfer of myristate from Nmt to acceptor proteins, the genes for hNmt and v-Src were co- expressed, and myristylation of pp60""" was assessed. We ob- served that mutation of Cys-169, His-218, or His-293 resulted in Nmts with greatly attenuated abilities to catalyze myristyl- ation of v-Src (Fig. 3). In contrast, mutations at Cys-214 and His-131 had relatively little effect on acyl product formation compared with the other conserved residue mutations, and thus appeared not to be critical to the transfer mechanism.

The effect of point mutations on Nmt activity was further supported by analysis of peptide myristylation activity in vitro. Fractions of bacterial lysate containing hNmt wild type, mu- tant, or histidine-tagged fusion protein were evaluated for their ability to transfer [:'H]myristyl-CoA to a peptide based on the N terminus of pp60'"""' (GSSKSKPKDPSY). As indicated in Table I, wild type, Cys-214, and His-131 proteins exhibited detectable levels of transferase activity. In agreement with results ob- tained by co-expressing Src with Nmt, mutation of residues at His-218 and His-293 also abrogated enzyme function in vitro.

kDa

zoo-+ 1 lh+ 97 - 66")

45-

32 +

22-

- 4- His-Nmt

1 < .1 ? , I

Vector alone HislO-hNmt

FIG. 2. Formation of a (myristylN-myristyl transferase) inter- mediate. N-terminal histidine tagged N-myristyl transferase, wild type and mutants, in PET 19b and BL21 DE3 bacteria were grown in LB media with 200 pg/ml ampicillin and 1 mhl IPTG in the presence of ["Hlmyristic acid. Cells were lysed in 100 p1 of SDS-PAGE sample buffer with 100 m>r dithiothreitol and sonicated. A,, = 0.147 (-10 pl) of each sample were analyzed by SDS-PAGE and fluorography. Exposure time was 6 days a t -80 "C. This experiment has been repeated 3 times with similar results. The band corresponding to the hNmt is marked by an arrow. An additional hand a t 116 kDa was sometimes observed, which may correspond to an Nmt dimer or Nmt bound to an E. coli protein. Upon excision of the 116 kDa band and re-electrophoresis, only the 50 kDa Nmt band was observed.

kDa

116- 97 + 6.3-

.,*.a*

45+ - f pp60v"rc

32 +

22") -= : NO No Src hNmt pp60vsrc+ hNmt

1 2 3 4 5 6 7 8 9

FIG. 3. Cotranslational ["HI-myristic acid incorporation into pp60""" (Src) catalyzed by hNmt, wild type, and mutants. hNmt (cu t ) in pETll (lane I), Src in modified pETl1 (lane 21, and Src plus hNmt (cut and mutants) in the same modified pETll (lanes 3-9) in BL21 DE3 bacteria were grown in the presence of ['Hlmyristic acid. Cells were lysed, and A,,, = 0.147(-10 pl) of each sample was analyzed by SDS-PAGE and fluorography. Exposure time was 13 days a t -80 "C. This experiment has been repeated 4 times with similar results.

Nmt with the C169S mutation showed marked reduction in activity compared with wild type and C214S. The residual level of activity of C169S may indicate that serine can substitute to a lesser degree for cysteine. Thus, a n ester acyl-enzyme inter- mediate may be able to function in the transfer mechanism, albeit at reduced efficiency compared with the thioester. Fi- nally, a caveat to any mutagenesis study is the possibility that some of the mutations might cause destabilization of overall protein structure.

Human N-Myristyl Bansferase Can Be Induced to Express a t Only Low Levels in Bacteria-The hNmt encoding wild type, mutant, and histidine-tagged fusion proteins could a t best be expressed at less than 100 ng/ml in BL21 DE3 and HMS174

Human N-Myristyl Transferase Active Site

DE3 bacteria under a T7 promotor. These results are consistent with a recent report (14). The hNmt gene (416 codons) contains 25 codons (6%) not normally used and another 49 codons (12%) used less than 10% of the time in translated E. coli genes (20). Poor codon usage by hNmt for E. coli is suggested as one prob- able, if not primary, cause for observed low expression levels.

Attempts to bind HislO-hNmts to a nickel chelating column were not successful. We therefore subcloned hNmt cDNA into the pCMV5 expression vector and transfected mammalian COS cells. High levels of protein production were observed by SDS- PAGE and Coomassie staining. A single 48-kDa hNmt protein was readily detected in COS cells overexpressing hNmt, based on Western blot with antipeptide antibodies against the hNmt sequence PDKDNIRQ (amino acids 60-67) (data not shown). A similar Western blot of E. coli lysates containing hNmt was negative, indicating that expression levels of hNmt in E. coli were below the threshold detection level of these antibodies.

Despite the difficulties encountered in directly quantitating Nmt levels in E. coli, several lines of evidence indicate that wild type and mutant forms of Nmt were expressed at equivalent levels. First, when bacteria were labeled with ~"S]SO, and lysed under nondenaturing conditions (20 mm Tris, pH 8.0, lysozyme), a band at approximately 50 kDa of comparable in- tensity was observed in wild type and mutant HislO-Nmt-ex-

TABLE I I n vitro incorporation of L3Hjmyristic acid into a Src peptide by

human N-myristyl transferase Bacteria containing the indicated Nmt construct were lysed, and

cytosolic fractions analyzed in vitro for Nmt activity, based on transfer of ['Hlmyristyl-CoA to a Src acceptor peptide. Activity measurements are the average of duplicate determinations.

""

Nmt construct Percent of wild type activity

"" ~

1. None 2.

0 2 1.0

3. (2169s 100 r 0.8 16 2 1.4

4. C214S 5. H131N

37 r 2.7 70 r 3.2

6. H171N 7. H218N

30 r 0.2 0 r 1.0

8. H293N 0 -c 1.0

Wild type

t - 4 (Iris 17 1 ?) H

f ?.) 0

I Fatty Acid,

pressing bacteria but not in bacteria containing empty vector (data not shown). Second, by Coomassie staining, all bacteria containing the Nmt and Src co-expression plasmid expressed equivalent amounts of pp60"-""' protein (data not shown). Since src and nmt genes are contained in the same plasmid and expressed under control of the same promoter, it is likely that the plasmids were present in comparable levels in equivalent amounts of bacteria. Finally, in vitro coupled transcription/ translation in rabbit reticulocyte lysates using the co-expres- sion vectors as cDNA templates revealed that both pp60"-""' and Nmt produced comparable amounts of translated protein (data not shown). Expression of hNmt in a eukaryotic system is ap- parently more favorable than in E. coli.

A Working Model for the Active Site of hNmt-The results presented in Figs. 2 and 3 are consistent with a model in which Cys-169 is the site of a covalent attachment of myristate to the enzyme. Histidine 171 would serve as a general acidibase and Histidine 293 as a specific acidibase needed for acyl-enzyme intermediate formation. A specific base (His-293) could enhance the cysteine thiol nucleophilicity for attack on the carbonyl of the myristyl CoA, while a general acid (His-171) would help relieve charge build-up on the carbonyl oxygen as it changes from a sp2 to a sp3 hybridization state (Fig. 4). A tetrahedral intermediate of myristate, CoA, and cysteine 169 would then decompose to acylated cysteine and CoA. To minimize the tran- sition state energy of this decomposition, a general base (His- 171) could assist in the rehybridization of the nascent carbonyl oxygen from a sp3 to a sp2 state, while a general acid (His-293) could aid the transition of the CoA sulfide from a thioester to a thiol. Carboxylic groups from spacially proximate aspartic and glutamic acid residues may also cooperate with the histidines to optimize acidibase properties (21).

Two possible roles can be postulated for the histidine at 218. First, His-218 may act as an acid or base needed for the trans- fer of the acyl group from the acyl-enzyme intermediate to a polypeptide substrate. The His-218 could serve as a base to deprotonate the amino group of the polypeptide N-terminal glycine to assist in its nucleophilic attack on the acyl-enzyme intermediate, or it may function as an acidibase to decompose the tetrahedral intermediate arising from such an attack (Fig.

n

0

H

a I - Fatly Acld 0 Acid

(His 218 ?) A c i d O H Fatty Acid Cys 169

Enzvml , Tetrahedral Intermediate Acyl Product and Decomposition Enzyme

FIG. 4. Working Model of hNmt Active Site. Proposed mechanism of hNmt active site with probable and possible roles of amino acids based on myristate binding and transfer studies of mutants.

30892 Human N-Myristyl Transferase Active Site

4). Alternatively, His-218 may be critical to a binding interac- tion of the nascent polypeptide. Mutation of His-218 could be deleterious to peptide substrate binding rather than to acyl transfer. By analogy to Nmtlp H221, which is noted below, the hNmt His-218 is expected to be on the surface of the enzyme upon initial myristyl-Coh approach while hNmt His-293 could be in a cleft with Cys-169.

It is expected that homologous cysteine and histidine residues found in the other N-myristyl transferases, such as Cys-172 in yeast Nmtlp, will serve identical roles in their myristyl transfer mechanisms. The dispensibility of the cysteine at 214 of hNmt to the transfer mechanism may explain why efforts to defini- tively identify the involvement of cysteine in the enzyme mech- anism of Nmtlp have been elusive (13). The active site cysteine is probably poorly accessible to chemical modification compared with the other conserved cysteine, Cys-217 in Nmtlp, which is likely to be near the protein surface as evidenced by the ability of chymotrypsin to cut Nmtlp between Thr-220 and His-221 (10). Binding offatty acyl-CoAto Nmt may be required to trigger access to the active site cysteine (Cys-169, hNmt; Cys-172, Nmtlp) for acyl-enzyme intermediate formation. Notably, no in- sertions or deletions in the number of amino acids in the linear polypeptide sequence are found between the conserved active site residues identified in these studies (Fig. 1). The conserved linear relationship between these residues may be reflected in a conserved three dimensional relationship.

Conclusions-The development of a working model at the molecular level for N-myristyl transferase will be of use in the design of regulators of N-myristyl transferase function. Our proposed model is consistent with the established enzyme mechanism and complements the fatty acid binding model de- veloped from substrate structure-activity studies (22-25). In the absence of definitive x-ray data of enzyme with bound sub- strates, such models serve as the best source on which to base future studies. The use of cysteines and histidines in the trans- fer mechanism is reminiscent of cysteine proteases (261, and may give clues to the source(s) from which N-myristyl trans- ferase has evolved.

Acknowledgments-We thank Mohanie Ramkishun and Raisa Louft-Nisenbaum for technical assistance, Claudette Bryant for secretarial assistance, and Luc Berthiaume and Cathy Sigal for valu- able discussions.

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