Site-directed mutation of conserved cysteine residues does not inactivate the Streptococcus pyogenes hyaluronan synthase.

Hyaluronan synthase (HAS), the enzyme responsible for the production of hyaluronic acid (HA), is a well-conserved membrane-bound protein in both prokaryotes and eukaryotes. This enzyme performs at least six discrete functions in producing a heterodisaccharide polymer of several million molecular weight and extruding it from the cell. Among the conserved motifs and domains within the Class I HAS family are four cysteine residues. Cysteines in many proteins are important in establishing and maintaining tertiary structure or in the coordination of catalytic functions. In the present study we utilized a combination of site-directed mutagenesis, chemical labeling, and kinetic analyses to determine the importance of specific Cys residues for catalysis and structure of the HA synthase from Streptococcus pyogenes (spHAS). The enzyme activity of spHAS was partially inhibited by cysteine-reactive chemical reagents such as N-ethylmaleimide. Quantitation of the number of Cys residues modified by these reagents, using MALDI-TOF mass spectrometry, demonstrated that there are no stable disulfide bonds in spHAS. The six Cys residues of spHAS were then mutated, individually and in various combinations, to serine or alanine. The single Cys-mutants were all kinetically similar to the wild-type enzyme in terms of their V(max) and K(m) values for HA synthesis. The Cys-null mutant, in which all Cys residues were mutated to alanine, retained approximately 66% of wild-type activity, demonstrating that despite their high degree of conservation within the HAS family, Cys residues are not absolutely necessary for HA biosynthesis by the spHAS enzyme.     

Mutation of conserved tryptophan residues at the dimer interface of Staphylococcus aureus nitric oxide synthase

Nitric oxide synthases (NOSs) share two invariant tryptophan residues within a conserved helical lariat that is part of the pterin-binding site and dimer interface. We mutated Staphylococcus aureus NOS Trp-314 (to alanine, phenylalanine, tyrosine and histidine) and Trp-316 (to alanine, phenylalanine and tyrosine) and characterized the effects of mutation on heme environment, quaternary structure, enzymatic activity, and substrate affinity. With arginine present, all saNOS variants bound heme with native thiolate ligation, formed high spin ferric complexes and were dimeric. All variants catalyze the peroxide-dependent oxidation of N-hydroxy-l-arginine, at rates from 10% to 55% of wild type activity. Arginine-free proteins are dimeric with the exception of W314A. Arginine affinity for all variants decreases with increasing temperature between 15 and 42 °C but is precipitous for position-314 variants. Previous structural and biophysical characterization of NOS oxygenase domains demonstrated that the protein can exist in either a tight or loose conformation, with the former corresponding to the active state of the protein. In the position-314 variants it is likely that the loose conformation is favoured, owing to the loss of a hydrogen bond between the indole side chain and the polypeptide backbone of the helical lariat.     

Mutation of conserved polar residues in the transmembrane domain of the proton-pumping pyridine nucleotide transhydrogenase of Escherichia coli

The pyridine nucleotide transhydrogenase carries out transmembrane proton translocation coupled to transfer of a hydride ion equivalent between NAD+ and NADP+. Previous workers (E. Holmberg et al. Biochemistry 33, 7691-7700, 1994; N. A. Glavas et al. Biochemistry 34, 7694-7702, 1995) had examined the role in proton translocation of conserved charged residues in the transmembrane domain. This study was extended to examine the role of conserved polar residues of the transmembrane domain. Site-directed mutagenesis of these residues did not produce major effects on hydride transfer or proton translocation activities except in the case of betaAsn222. Most mutants of this residue were drastically impaired in these activities. Three phenotypes were recognized. In betaN222C both activities were impaired maximally by 70%. The retention of proton translocation indicated that betaAsn222 was not directly involved in proton translocation. In betaN222H both activities were drastically reduced. Binding of NADP+ but not of NADPH was impaired. In betaN222R, by contrast, NADP+ remained tightly bound to the mutant transhydrogenase. It is concluded that betaAsn222, located in a transmembrane alpha-helix, is part of the conformational pathway by which NADP(H) binding, which occurs outside of the transmembrane domain, is coupled to proton translocation. Some nonconserved or semiconserved polar residues of the transmembrane domain were also examined by site-directed mutagenesis. Interaction of betaGlu124 with the proton translocation pathway is proposed.     

Substitution of conserved residues within the active site alters the cleavage religation equilibrium of DNA topoisomerase I

Eukaryotic DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of camptothecin (CPT). Mutation of conserved residues in close proximity to the active site tyrosine (Tyr(727) of yeast Top1p) alters the DNA cleavage religation equilibrium, inducing drug-independent cell lethality. Previous studies indicates that yeast Top1T722Ap and Top1N726Hp cytotoxicity results from elevated levels of covalent enzyme-DNA intermediates. Here we show that Top1T722Ap acts as a CPT mimetic by exhibiting reduced rates of DNA religation, whereas increased Top1N726Hp.DNA complexes result from elevated DNA binding and cleavage. We also report that the combination of the T722A and N726H mutations in a single protein potentiates the cytotoxic action of the enzyme beyond that induced by co-expression of the single mutants. Moreover, the addition of CPT to cells expressing the double top1T722A/N726H mutant did not enhance cell lethality. Thus, independent alterations in DNA cleavage and religation contribute to the lethal phenotype. The formation of distinct cytotoxic lesions was also evidenced by the different responses induced by low levels of these self-poisoning enzymes in isogenic strains defective for the Rad9 DNA damage checkpoint, processive DNA replication, or ubiquitin-mediated proteolysis. Substitution of Asn(726) with Phe or Tyr also produces self-poisoning enzymes, implicating stacking interactions in the increased kinetics of DNA cleavage by Top1N726Hp and Top1N726Fp. In contrast, replacing the amide side chain of Asn(726) with Gln renders Top1N726Qp resistant to CPT, suggesting that the orientation of the amide within the active site is critical for effective CPT binding.     

Roles of conserved methionine residues in tobacco acetolactate synthase

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. ALS is the target of several classes of herbicides, including the sulfonylureas, the imidazolinones, and the triazolopyrimidines. The conserved methionine residues of ALS from plants were identified by multiple sequence alignment using ClustalW. The alignment of 17 ALS sequences from plants revealed 149 identical residues, seven of which were methionine residues. The roles of three well-conserved methionine residues (M350, M512, and M569) in tobacco ALS were determined using site-directed mutagenesis. The mutation of M350V, M512V, and M569V inactivated the enzyme and abolished the binding affinity for cofactor FAD. Nevertheless, the secondary structure of each of the mutants determined by CD spectrum was not affected significantly by the mutation. Both M350C and M569C mutants were strongly resistant to three classes of herbicides, Londax (a sulfonylurea), Cadre (an imidazolinone), and TP (a triazolopyrimidine), while M512C mutant did not show a significant resistance to the herbicides. The mutant M350C was more sensitive to pH change, while the mutant M569C showed a profile for pH dependence activity similar to that of wild type. These results suggest that M512 residue is likely located at or near the active site, and that M350 and M569 residues are probably located at the overlapping region between the active site and a common herbicide binding site.     

Activation of MEK family kinases requires phosphorylation of two conserved Ser/Thr residues.

MEK is a family of dual specific protein kinases which activate the extracellular signal-regulated kinases by phosphorylation of threonine and tyrosine residues. MEK itself is activated via serine phosphorylation by upstream activator kinases, including c-raf, mos and MEK kinase. Here, we report the activation phosphorylation sites of human MEK1 and yeast STE7 kinase as determined by a combination of biochemical and genetic approaches. In human MEK1, substitution of either serine residue 218 or 222 with alanine completely abolished its activation by epidermal growth factor-stimulated Swiss 3T3 cell lysates or immunoprecipitated c-raf, suggesting that both serine residues are required for MEK1 activation. Phosphopeptide analysis demonstrated that serine residues 218 and 222 of human MEK1 are the primary sites for phosphorylation by c-raf. These two serine residues are highly conserved in all members of the MEK family, including the yeast STE7 gene product, a MEK homolog in the yeast mating pheromone response pathway. Mutation of the corresponding residues in STE7 completely abolished the biological functions of this gene. These data demonstrate that MEK is activated by phosphorylation of two adjacent serine/threonine residues and this activation mechanism is conserved in the MEK family kinases.     

Mutation of active site residues of insulin-degrading enzyme alters allosteric interactions

The active site glutamate (Glu(111)) and the active site histidine (His(112)) of insulin-degrading enzyme (IDE) were mutated. These mutant enzymes exhibit, in addition to a large decrease in catalytic activity, a change in the substrate-velocity response from a sigmoidal one seen with the native enzyme (Hill coefficient > 2), to a hyperbolic response. With 2-aminobenzoyl-GGFLRKHGQ-N-(2,4-dinitrophenyl)ethylenediamine as substrate, ATP and triphosphate increase the reaction rate of the wild type enzyme some 50-80-fold. This effect is dampened with glutamate mutants to no effect or less than a 3-fold increase in activity and changed to inhibition with the histidine mutants. Sedimentation equilibrium shows the IDE mutants exhibit a similar oligomeric distribution as the wild type enzyme, being predominantly monomeric, with triphosphate having little if any effect on the oligomeric state. Triphosphate did induce aggregation of many of the IDE mutants. Thus, the oligomeric state of IDE does not correlate with kinetic properties. The His(112) mutants were shown to bind zinc, but with a lower affinity than the wild type enzyme. The glutamate mutants displayed an altered cleavage profile for the peptide beta-endorphin. Wild type IDE cleaved beta-endorphin at Leu(17)-Phe(18) and Phe(18)-Lys(19), whereas the glutamate mutants cleaved at these sites, but in addition at Lys(19)-Asn(20) and at Met(5)-Thr(6). Thus, active site mutations of IDE are suggested to not only reduce catalytic activity but also cause local conformational changes that affect the allosteric properties of the enzyme.     

Mutation of essential catalytic residues in pig citrate synthase.

Two amino acid residues, His274 and Asp375, were replaced singly in the active site of pig citrate synthase (PCS) with Gly274, Arg274, Gly375, Asn375, Glu375, and Gln375. The nonmutant protein and the mutant proteins were expressed in and purified from Escherichia coli, and the effects of these amino acid substitutions on the overall reaction rate and conformation of the PCS protein were studied by initial velocity and full time course kinetic analysis, behavior during affinity column chromatography, and monoclonal antibody reactivity. Native and mutant proteins purified similarly had a subunit molecular weight of 50,000 and were homologous when examined with 10 independent a-PCS monoclonal IgGs or with a polyclonal anti-PHCS serum. No activity was detected for Asn375 or Gln375. The kcats of the other purified mutant proteins, however, were decreased by about 10(3) compared to the nonmutant enzyme activity. The Km for oxalacetate was decreased 10-fold in the Glu375 protein and was reduced by half in Gly274 and Arg274 PCSs, while the Km for acetyl-CoA was decreased 2-3-fold in Gly274, Arg274, and Gln375 PCSs. A mechanism is proposed that electrostatically links His274 and Asp375.     

Analysis of the two active sites of the hyaluronan synthase and the chondroitin synthase of Pasteurella multocida

Type A Pasteurella multocida produces a hyaluronan (HA) capsule to enhance infection. The 972-residue HA synthase, pmHAS, polymerizes the linear HA polysaccharide composed of alternating beta3N-acetylglucosamine (GlcNAc)-beta4glucuronic acid (GlcUA). We demonstrated previously that pmHAS possesses two independent glycosyltransferase sites. Here we further define the sites and putative motifs. Deletion of residues 1-117 does not affect HA polymerizing activity. The carboxyl-terminal boundary of the GlcUA-transferase resides within residues 686-703. Both transferase sites contain a DXD motif essential for HA synthase activity. D247N or D249N mutants possessed only GlcUA-transferase activity, whereas D527N or D529N mutants possessed only GlcNAc-transferase activity, further confirming our assignment of the two active sites within the synthase polypeptide. A potential role of the DXD motif in substrate binding was supported by experiments utilizing high UDP-sugar concentrations that partially rescued the activity of certain mutants. The WGGED sequence motif is involved in GlcNAc-transferase activity because mutants with substitutions at E369 or D370 possessed only GlcUA-transferase activity. Type F P. multocida synthesizes an unsulfated chondroitin (beta3GalNAc-beta4GlcUA) capsule. A chimeric enzyme consisting of residues 1-427 of pmHAS and residues 421-704 of pmCS, the homologous chondroitin synthase, was an active HA synthase. The converse chimeric enzyme consisting of residues 1-420 of pmCS and residues 428-703 of pmHAS was a functional chondroitin synthase. Analyses of a panel of pmHAS/pmCS chimeric enzymes identified a 44-residue region, corresponding to pmHAS residues 225-265, involved in UDP-hexosamine selectivity. Overall, these findings further support the model of two independent transferase sites within a single polypeptide.     

Mutational analysis of conserved outer sphere arginine residues of chalcone synthase

Chalcone synthase (CHS), a key enzyme in flavonoid biosynthesis, catalyses sequential decarboxylative condensations of p-coumaroyl-CoA with three malonyl-CoA molecules and cyclizes the resulting tetraketide intermediate to produce chalcone. Phenylglyoxal, an Arg selective reagent, was found to inactivate the enzyme, although no Arg is found at the active site. Conserved, non-active site Arg residues of CHS were individually mutated and the results were discussed in the context of the 3D structure of CHS. Arg199 and Arg350 were shown to provide important interactions to maintain the structural integrity and foldability of the enzyme. Arg68, Arg172 and Arg328 interact with highly conserved Gln33/Phe215, Glu380 and Asp311/Glu314, respectively, thus helping position the catalytic Cys-His-Asn triad and the (372)GFGPG loop in correct topology at the active site. In particular, a mutation of Arg172 resulted in selective impairment in the cyclization activities of CHS and stilbene synthase, a related enzyme that catalyses a different cyclization of the same tetraketide intermediate. These Arg residues and their interactions are well conserved in other enzymes of the CHS superfamily, suggesting that they may serve similar functions in other enzymes. Mutations of Arg68 and Arg328 had been found in mutant plants that showed impaired CHS activity.     

Mutation of conserved histidines alters tertiary structure and nanomechanics of consensus ankyrin repeats

The conserved TPLH tetrapeptide motif of ankyrin repeats (ARs) plays an important role in stabilizing AR proteins, and histidine (TPLH)-to-arginine (TPLR) mutations in this motif have been associated with a hereditary human anemia, spherocytosis. Here, we used a combination of atomic force microscopy-based single-molecule force spectroscopy and molecular dynamics simulations to examine the mechanical effects of His → Arg substitutions in TPLH motifs in a model AR protein, NI6C. Our molecular dynamics results show that the mutant protein is less mechanically stable than the WT protein. Our atomic force microscopy results indicate that the mechanical energy input necessary to fully unfold the mutant protein is only half of that necessary to unfold the WT protein (53 versus 106 kcal/mol). In addition, the ability of the mutant to generate refolding forces is also reduced. Moreover, the mutant protein subjected to cyclic stretch-relax measurements displays mechanical fatigue, which is absent in the WT protein. Taken together, these results indicate that the His → Arg substitutions in TPLH motifs compromise mechanical properties of ARs and suggest that the origin of hereditary spherocytosis may be related to mechanical failure of ARs.     

Clarifying the catalytic roles of conserved residues in the amidase signature family

Fatty acid amide hydrolase (FAAH) is a mammalian integral membrane enzyme responsible for the hydrolysis of a number of neuromodulatory fatty acid amides, including the endogenous cannabinoid anandamide and the sleep-inducing lipid oleamide. FAAH belongs to a large class of hydrolytic enzymes termed the "amidase signature family," whose members are defined by a conserved stretch of approximately 130 amino acids termed the "amidase signature sequence." Recently, site-directed mutagenesis studies of FAAH have targeted a limited number of conserved residues in the amidase signature sequence of the enzyme, identifying Ser-241 as the catalytic nucleophile and Lys-142 as an acid/base catalyst. The roles of several other conserved residues with potentially important and/or overlapping catalytic functions have not yet been examined. In this study, we have mutated all potentially catalytic residues in FAAH that are conserved among members of the amidase signature family, and have assessed their individual roles in catalysis through chemical labeling and kinetic methods. Several of these residues appear to serve primarily structural roles, as their mutation produced FAAH variants with considerable catalytic activity but reduced expression in prokaryotic and/or eukaryotic systems. In contrast, five mutations, K142A, S217A, S218A, S241A, and R243A, decreased the amidase activity of FAAH greater than 100-fold without detectably impacting the structural integrity of the enzyme. The pH rate profiles, amide/ester selectivities, and fluorophosphonate reactivities of these mutants revealed distinct catalytic roles for each residue. Of particular interest, one mutant, R243A, displayed uncompromised esterase activity but severely reduced amidase activity, indicating that the amidase and esterase efficiencies of FAAH can be functionally uncoupled. Collectively, these studies provide evidence that amidase signature enzymes represent a large class of serine-lysine catalytic dyad hydrolases whose evolutionary distribution rivals that of the catalytic triad superfamily.     

Alteration of polysaccharide size distribution of a vertebrate hyaluronan synthase by mutation

Hyaluronan (HA) is a nonsulfated glycosaminoglycan that has long been known to play structural roles in vertebrates. Recently, it has become increasingly obvious that this linear polysaccharide has many more uses than simply scaffolding or space filler. HA has been found to be involved in development, cell signaling, cell motility, and metastasis. These roles are often dictated by the length of the HA polymer, which can vary from a few to about 10,000 sugar residues in length. Three distinct isoforms of HA synthase exist in mammals. It has been shown previously by others that each isoform produces HA that differs in size distribution, but the regulatory mechanism is not yet known. Mutations have been described that alter the size distribution of the HA produced by the streptococcal HA synthases. We show that by mutating one particular amino acid residue of a vertebrate HA synthase, depending on the introduced side chain, the size of HA produced can be either reduced or increased. We postulate that several cysteine residues and a serine residue may be involved in binding directly or indirectly to the nascent HA chain. These data support the theory that the relative strength of the interaction between the catalyst and the polymer may be a major factor in HA size control.     

The influence of conserved aromatic residues in 3-hydroxy-3-methylglutaryl-CoA synthase

In order to evaluate the potential contribution of conserved aromatic residues to the hydrophobic active site of 3-hydroxy-3-methylglutaryl-CoA synthase, site-directed mutagenesis was employed to produce Y130L, Y163L, F204L, Y225L, Y346L, and Y376L proteins. Each mutant protein was expressed at levels comparable with wild-type enzyme and was isolated in highly purified form. Initial kinetic characterization indicated that F204L exhibits a substantial (>300-fold) decrease in catalytic rate (kcat). Upon modification with the mechanism-based inhibitor, 3-chloropropionyl-CoA, or in formation of a stable binary complex with acetoacetyl-CoA, F204L exhibits binding stoichiometries comparable with wild-type enzyme, suggesting substantial retention of active site integrity. Y130L and Y376L exhibit inflated values (80- and 40-fold, respectively) for the Km for acetyl-CoA in the acetyl-CoA hydrolysis partial reaction; these mutants also exhibit an order of magnitude decrease in kcat. Formation of the acetyl-S-enzyme reaction intermediate by Y130L, F204L, and Y376L proceeds slowly in comparison with wild-type enzyme. However, solvent exchange into the thioester carbonyl oxygen of these acetyl-S-enzyme intermediates is not slow in comparison with previous observations for D159A and D203A mutants, which also exhibit slow acetyl-S-enzyme formation. The magnitude of the differential isotope shift upon exchange of H218O into [13C]acetyl-S-enzyme suggests a polarization of the thioester carbonyl and a reduction in bond order. Such an effect may substantially contribute to the upfield 13C NMR shift observed for [13C]acetyl-S-enzyme. The influence on acetyl-S-enzyme formation, as well as observed kcat (F204L) and Km (Y130L; Y376L) effects, implicate these invariant residues as part of the catalytic site. Substitution of phenylalanine (Y130F, Y376F) instead of leucine at residues 130 and 376 diminishes the effects on catalytic rate and substrate affinity observed for Y130L and Y376L, underscoring the influence of aromatic side chains near the active site.     

The hyaluronan synthase catalyzes the synthesis and membrane translocation of hyaluronan

Hyaluronan (HA), an extracellular linear polysaccharide of alternating N-acetyl-glucosamine and glucuronic acid residues, is ubiquitously expressed in vertebrates, where it affects a broad spectrum of physiological processes, including cell adhesion, migration and differentiation. The HA polymer is synthesized on the cytosolic side of the cell membrane by the membrane-embedded hyaluronan synthase (HAS). However, the process by which the extremely hydrophilic HA polymer is translocated across the membrane is unknown to date. The bacterial HAS from Streptococcus equisimilis (Se) shares a similar transmembrane topology and significant sequence identity with human HASs and likely synthesizes HA by the same mechanism. We demonstrate that the Se-HAS is both necessary and sufficient to translocate HA in a reaction that is tightly coupled to HA elongation. The purified Se-HAS is reconstituted into proteoliposomes (PLs) where it synthesizes and translocates HA. In vitro synthesized, high-molecular-weight HA remains tightly associated with the intact PLs in sedimentation experiments. Most importantly, the newly formed HA is protected from enzymatic degradation by hyaluronidase unless the PLs are solubilized with detergent, thereby demonstrating that HA is translocated into the lumen of the vesicle. In addition, we show that HA synthesis and translocation are spatially coupled events, which allow HA synthesis even in the presence of a large excess of HA-degrading enzyme. The coupled synthesis and membrane translocation of a biopolymer represents a novel membrane translocation mechanism and is likely applicable to the synthesis of some of the most abundant biopolymers, including chitin and cellulose.     

Topological analyses of the L-lysine exporter LysO reveal a critical role for a conserved pair of intramembrane solvent-exposed acidic residues

LysO, a prototypical member of the LysO family, mediates export of L-lysine (Lys) and resistance to the toxic Lys antimetabolite, L-thialysine (Thl) in Escherichia coli. Here, we have addressed unknown aspects of LysO function pertaining to its membrane topology and the mechanism by which it mediates Lys/Thl export. Using substituted cysteine (Cys) accessibility, here we delineated the membrane topology of LysO. Our studies support a model in which both the N- and C-termini of LysO are present at the periplasmic face of the membrane with a transmembrane (TM) domain comprising eight TM segments (TMSs) between them. In addition, a feature of intramembrane solvent exposure in LysO is inferred with the identification of membrane-located solvent-exposed Cys residues. Isosteric substitutions of a pair of conserved acidic residues, one E233, located in the solvent-exposed TMS7 and the other D261, in a solvent-exposed intramembrane segment located between TMS7 and TMS8, abolished LysO function in vivo. Thl, but not Lys, elicited proton release in inside-out membrane vesicles, a process requiring the presence of both E233 and D261. We postulate that Thl may be exported in antiport with H⁺ and that Lys may be a low-affinity export substrate. Our findings are compatible with a physiological scenario wherein in vivo LysO exports the naturally occurring antimetabolite Thl with higher affinity over the essential cellular metabolite Lys, thus affording protection from Thl toxicity and limiting wasteful export of Lys.     

Point mutations in two conserved glycine residues within the integral membrane protein FhuB affect iron(II) hydroxamate transport

Summary A region of substantial homology, comprising 32 amino acids around a highly conserved glycine residue, is located near the C-terminal ends of the hydrophobic Fhu, Fec, Fep, Fat, and Btu transport proteins involved in the uptake of ferrisiderophores and vitamin B₁₂ into Escherichia coli and Vibrio anguillarum. Furthermore, a region similar in location and sequence containing an invariant glycine at an equivalent position was identified in the hydrophobic component of all other periplasmic binding protein-dependent (PBT) systems. In the FhuB protein, which is twice the size of the other PBT-related inner membrane proteins and which displays an internal homology, two conserved glycine residues are present. Alteration of Gly at positions 226 and 559 to Ala, Val, or Glu reduced iron(III) hydroxamate uptake, suggesting that this homologous region may play a general role in the mechanism of PBT-dependent transport.     

E. coli cardiolipin synthase: Function of N-terminal conserved residues

The E. coli cls open reading frame (ORF) predicts a 54.8 kDa polypeptide, whereas mature cardiolipin (CL) synthase is 46 kDa. The N-terminal region extending to residue 60 contains several conserved residues but is not essential for enzyme activity. A deletion mutant that is missing residues 2-60 produces a fully active protein. These findings raise the question of why several residues in a region that is not required for enzyme activity are conserved. Recombinant DNA technology was used to introduce an EYMPE epitope (EE) tag into the interior of CL synthase. The EE tagged polypeptide retained the biological properties of wild type CL synthase, including full enzymatic activity. Site-directed mutagenesis was used to alter conserved residues in the N-terminal region. An EE tagged CL synthase in which Leu-7 and Val-8 were both replaced by Ser residues retains in vitro activity but loses most of its in vivo activity. Furthermore, the mutant protein has a higher apparent molecular mass than its parent protein. Taken together, these findings suggest that conserved residues L7 and V8 play a role in polypeptide processing, topology, or both.