Novel alkyl alkanethiolsulfonate sulfhydryl reagents. Modification of derivatives ofl-cysteine

A simple, general scheme for the synthesis of sulfhydryl-specific alkyl alkanethiolsulfonate (RSSO₂R′) reagents where R′ is methyl, has been developed. Two new reagents, methyl aminoethanethiolsulfonate (2) and methyl benzylthiolsulfonate (3) were synthesized. These were used to modify stoichiometrically and selectively under mild conditions the sulfhydryl groups ofN-acetyl-l-cysteine ethyl ester (4),N-acetyl-l-cysteinep-nitroanilide (7), glutathione, and the A chain of bovine insulin. The corresponding β-S-(β-aminoethanethiol) and β-S-(benzylthiol) derivatives ofl-cysteine and of the peptides were afforded. The characteristics and significance of these reactions and products are discussed.     

Simultaneous electrochemical determination of l-cysteine and l-cysteine disulfide at carbon ionic liquid electrode

A linear sweep voltammetric method is used for direct simultaneous determination of l-cysteine and l-cysteine disulfide (cystine) based on carbon ionic liquid electrode. With carbon ionic liquid electrode as a high performance electrode, two oxidation peaks for l-cysteine (0.62 V) and l-cysteine disulfide (1.3 V) were observed with a significant separation of about 680 mV (vs. Ag/AgCl) in phosphate buffer solution (pH 6.0). The linear ranges were obtained as 1.0–450 and 5.0–700 μM and detection limits were estimated to be 0.298 and 4.258 μM for l-cysteine and l-cysteine disulfide, respectively. This composite electrode was applied for simultaneous determination of l-cysteine and l-cysteine disulfide in two real samples, artificial urine and nutrient broth. Satisfactory results were obtained which clearly indicate the applicability of the proposed electrode for simultaneous determination of these compounds in complex matrices.     

Functional cardiovascular action of l-cysteine microinjected into pressor sites of the rostral ventrolateral medulla of the rat

The endogenous sulfur-containing amino acid l-cysteine injected into the cerebrospinal fluid space of the cisterna magna increases arterial blood pressure (ABP) and heart rate (HR) in the freely moving rat. The present study examined (1) cardiovascular responses to l-cysteine microinjected into the rostral ventrolateral medulla (RVLM), where a group of neurons regulate activities of cardiovascular sympathetic neurons and (2) involvement of ionotropic excitatory amino acid (iEAA) receptors in response. In the RVLM of urethane-anesthetized rats accessed ventrally and identified with pressor responses to l-glutamate (10 mM, 34 nl), microinjections of l-cysteine increased ABP and HR dose dependently (3–100 mM, 34 nl). The cardiovascular responses to l-cysteine (30 mM) were not attenuated by a prior injection of either antagonist alone, MK801 (20 mM, 68 nl) for the NMDA type of iEAA receptors, or CNQX (2 mM) for the non-NMDA type. However, inhibition of both NMDA and non-NMDA receptors with additional prior injection of either antagonist completely blocked those responses to l-cysteine. The results indicate that l-cysteine has functional cardiovascular action in the RVLM of the anesthetized rat, and the responses to l-cysteine involve both NMDA and non-NMDA receptors albeit in a mutually exclusive parallel fashion. The findings may suggest endogenous roles of l-cysteine indirectly via iEAA receptors in the neuronal network of the RVLM for cardiovascular regulation in physiological and pathological situations.     

Characterization of a recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus that isomerizes l-fucose, d-arabinose, d-altrose, and l-galactose

A recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus was purified as a single 68 kDa band with an activity of 76 U mg^?1. The molecular mass of the native enzyme was 204 kDa as a trimer. The maximum activity for l-fucose isomerization was at pH 7 and 75°C in the presence of 1 mM Mn²⁺. Its half-life at 70°C was 6.1 h. For aldose substrates, the enzyme displayed activity in decreasing order for l-fucose, with a k _cat of 11,910 min^?1 and a K _m of 140 mM, d-arabinose, d-altrose, and l-galactose. These aldoses were converted to the ketoses l-fuculose, d-ribulose, d-psicose, and l-tagatose, respectively, with 24, 24, 85, 55% conversion yields after 3 h.     

l-leucine, l-methionine, and l-phenylalanine share a Na+/K+-dependent amino acid transporter in shrimp hepatopancreas

Hepatopancreatic brush border membrane vesicles (BBMV), made from Atlantic White shrimp (Litopenaeus setiferus), were used to characterize the transport properties of ³H-l-leucine influx by these membrane systems and how other essential amino acids and the cations, sodium and potassium, interact with this transport system. ³H-l-leucine uptake by BBMV was pH-sensitive and occurred against transient transmembrane concentration gradients in both Na⁺- and K⁺-containing incubation media, suggesting that either cation was capable of providing a driving force for amino acid accumulation. ³H-l-leucine uptake in NaCl or KCl media were each three times greater in acidic pH (pH 5.5) than in alkaline pH (pH 8.5). The essential amino acid, l-methionine, at 20 mM significantly (p < 0.0001) inhibited the 2-min uptakes of 1 mM ³H-l-leucine in both Na⁺- and K⁺-containing incubation media. The residual ³H-l-leucine uptake in the two media were significantly greater than zero (p < 0.001), but not significantly different from each other (p > 0.05) and may represent an l-methionine- and cation-independent transport system. ³H-l-leucine influxes in both NaCl and KCl incubation media were hyperbolic functions of [l-leucine], following the carrier-mediated Michaelis–Menten equation. In NaCl, ³H-l-leucine influx displayed a low apparent K _M (high affinity) and low apparent J _max, while in KCl the transport exhibited a high apparent K _M (low affinity) and high apparent J _max. l-methionine or l-phenylalanine (7 and 20 mM) were competitive inhibitors of ³H-l-leucine influxes in both NaCl and KCl media, producing a significant (p < 0.01) increase in ³H-l-leucine influx K _M, but no significant response in ³H-l-leucine influx J _max. Potassium was a competitive inhibitor of sodium co-transport with ³H-l-leucine, significantly (p < 0.01) increasing ³H-l-leucine influx K _M in the presence of sodium, but having negligible effect on ³H-l-leucine influx J _max in the same medium. These results suggest that shrimp BBMV transport ³H-l-leucine by a single l-methionine- and l-phenylalanine-shared carrier system that is enhanced by acidic pH and can be stimulated by either Na⁺ or K⁺ acting as co-transport drivers binding to shared activator sites.     

Backbone and side-chain 1H, 15N and 13C assignment of apo- and imipenem-acylated l,d-transpeptidase from Bacillus subtilis

The d,d-transpeptidase activity of Penicillin Binding Proteins (PBPs) is essential to maintain cell wall integrity. PBPs catalyze the final step of the peptidoglycan synthesis by forming 4 → 3 cross-links between two peptide stems. Recently, a novel β-lactam resistance mechanism involving l,d-transpeptidases has been identified in Enterococcus faecium and Mycobacterium tuberculosis. In this resistance pathway, the classical 4 → 3 cross-links are replaced by 3 → 3 cross-links, whose formation are catalyzed by the l,d-transpeptidases. To date, only one class of the entire β-lactam family, the carbapenems, is able to inhibit the l,d-transpeptidase activity. Nevertheless, the specificity of this inactivation is still not understood. Hence, the study of this new transpeptidase family is of considerable interest in order to understand the mechanism of the l,d-transpeptidases inhibition by carbapenems. In this context, we present herein the backbone and side-chain ¹H, ¹⁵N and ¹³C NMR assignment of the l,d-transpeptidase from Bacillus subtilis (Ldt_Bs) in the apo and in the acylated form with a carbapenem, the imipenem.     

Enantioselective N-acetylation of 2-phenylglycine by an unusual N-acetyltransferase from Chryseobacterium sp.

The demand for d-2-phenylglycine used to synthesize semisynthetic antibiotics and pesticides is increasing. We have isolated a Chryseobacterium sp. that selectively transformed the l-form of racemic d,l-2-phenylglycine to (2S)-2-acetylamide-2-phenylacetic acid with a molar yield of 50 % and an enantiomer excess of >99.5 % under optimal culture conditions, consequently resulting in 99 % pure d-2-phenylglycine remaining in the culture. The enantioselective N-acetylation was catalyzed by an acetyl-CoA-dependent N-acetyltransferase whose synthesis was induced by l-2-phenylglycine. The enzyme differed from previously reported bacterial arylamine N-acetyltransferases in molecular mass and substrate specificity. The relative activity ratio of the enzyme with the substrates l-2-phenylglycine, d-2-phenylglycine, 2-(2-chlorophenyl)glycine, and 5-aminosalicylic acid (a good substrate of arylamine N-acetyltransferase) was 100:0:56.9:5.49, respectively. The biotransformation by the N-acetyltransferase-producing bacterium reported here could constitute a new preparative route for the enzymatic resolution of d,l-2-phenylglycine.     

l-Arabinose/d-galactose 1-dehydrogenase of Rhizobium leguminosarum bv. trifolii characterised and applied for bioconversion of l-arabinose to l-arabonate with Saccharomyces cerevisiae

Four potential dehydrogenases identified through literature and bioinformatic searches were tested for l-arabonate production from l-arabinose in the yeast Saccharomyces cerevisiae. The most efficient enzyme, annotated as a d-galactose 1-dehydrogenase from the pea root nodule bacterium Rhizobium leguminosarum bv. trifolii, was purified from S. cerevisiae as a homodimeric protein and characterised. We named the enzyme as a l-arabinose/d-galactose 1-dehydrogenase (EC 1.1.1.-), Rl AraDH. It belongs to the Gfo/Idh/MocA protein family, prefers NADP⁺ but uses also NAD⁺ as a cofactor, and showed highest catalytic efficiency (k _cat/K _m) towards l-arabinose, d-galactose and d-fucose. Based on nuclear magnetic resonance (NMR) and modelling studies, the enzyme prefers the α-pyranose form of l-arabinose, and the stable oxidation product detected is l-arabino-1,4-lactone which can, however, open slowly at neutral pH to a linear l-arabonate form. The pH optimum for the enzyme was pH 9, but use of a yeast-in-vivo-like buffer at pH 6.8 indicated that good catalytic efficiency could still be expected in vivo. Expression of the Rl AraDH dehydrogenase in S. cerevisiae, together with the galactose permease Gal2 for l-arabinose uptake, resulted in production of 18 g of l-arabonate per litre, at a rate of 248 mg of l-arabonate per litre per hour, with 86 % of the provided l-arabinose converted to l-arabonate. Expression of a lactonase-encoding gene from Caulobacter crescentus was not necessary for l-arabonate production in yeast.     

Protective effect of l-ascorbic acid on nickel induced pulmonary nitrosative stress in male albino rats

Nickel sulfate stimulates inducible nitric oxide synthase (i-NOS) and increases serum nitric oxide concentration by overproduction of reactive nitrogen species due to nitrosative stress. The present study was undertaken to assess possible protective role of l-ascorbic acid as an antioxidant against nickel induced pulmonary nitrosative stress in male albino rats. We studied the effect of the simultaneous treatment with l-ascorbic acid (50 mg/100 g b. wt.; orally) and nickel sulfate (2.0 mg/100 g b. wt.; i.p.) on nitric oxide synthesis by quantitative evaluation of serum i-NOS activities, serum and lung nitric oxide, l-ascorbic acid and protein concentrations of Wister strain male albino rats. We have further studied histopathological changes in lung tissue after nickel sulfate treatment along with simultaneous exposure of l-ascorbic acid. Nickel sulfate treatment significantly increased the serum i-NOS activity, serum and pulmonary nitric oxide concentration and decreased body weight, pulmonary somatic index, serum and lung l-ascorbic acid and protein concentration as compared to their respective controls. Histopathological changes induced by nickel sulfate showed loss of normal alveolar architecture, inflammation of bronchioles, infiltration of inflammatory cells and patchy congestion of alveolar blood vessels. The simultaneous administration of l-ascorbic acid and nickel sulfate significantly improved all the above biochemical parameters along with histopathology of lung tissues of rats receiving nickel sulfate alone. The study clearly showed a protective role of l-ascorbic acid against nickel induced nitrosative stress in lung tissues.     

Recombinant production and characterization of an N-acyl-d-amino acid amidohydrolase from Streptomyces sp. 64E6

N-Acyl-d-amino acid amidohydrolases (d-aminoacylases) are often used as tools for the optical resolution of d-amino acids, which are important products with applications in industries related to medicine and cosmetics. For this study, genes encoding d-aminoacylase were cloned from the genomes of Streptomyces spp. using sequence-based screening. They were expressed by Escherichia coli and Streptomyces lividans. Almost all of the cell-free extracts exhibit hydrolytic activity toward N-acetyl-(Ac-)d-Phe (0.05–6.32 μmol min^?1 mg^?1) under conditions without CoCl₂. Addition of 1 mM CoCl₂ enhanced their activity. Among them, the highest activity was observed from cell-free extracts prepared from S. lividans that possess the d-aminoacylase gene of Streptomyces sp. 64E6 (specific activities were, respectively, 7.34 and 9.31 μmol min^?1 mg^?1 for N-Ac-d-Phe and N-Ac-d-Met hydrolysis). Furthermore, when using glycerol as a carbon source for cultivation, the recombinant enzyme from Streptomyces sp. 64E6 was produced in 4.2-fold greater quantities by S. lividans than when using glucose. d-Aminoacylase from Streptomyces sp. 64E6 showed optimum at pH 8.0–9.0. It was stable at pH 5.5–9.0 up to 30 °C. The enzyme hydrolyzed various N-acetyl-d-amino acids that have hydrophobic side chains. In addition, the activity toward N-chloroacetyl-d-Phe was 2.1-fold higher than that toward N-Ac-d-Phe, indicating that the structure of N-acylated portion of substrate altered the activity.     

Preparation of eutectic substrate mixtures for enzymatic conversion of ATC to l-cysteine at high concentration levels

High concentration eutectic substrate solutions for the enzymatic production of l-cysteine were prepared. Eutectic melting of binary mixtures consisting of d,l-2-amino-Δ²-thiazoline-4-carboxylic acid (ATC) as a substrate and malonic acid occurred at 39 °C with an ATC mole fraction of 0.5. Formation of eutectic mixtures was confirmed using SEM, SEM–EDS, and XPS surface analyses. Sorbitol, MnSO₄, and NaOH were used as supplements for the enzymatic reactions. Strategies for sequential addition of five compounds, including a binary ATC mixture and supplements, during preparation of eutectic substrate solutions were established. Eutectic substrate solutions were stable for 24 h. After 6 h of enzymatic reactions, a 550 mM l-cysteine yield was obtained from a 670 mM eutectic ATC solution.     

Pressor response to l-cysteine injected into the cisterna magna of conscious rats involves recruitment of hypothalamic vasopressinergic neurons

The sulfur-containing non-essential amino acid l-cysteine injected into the cisterna magna of adult conscious rats produces an increase in blood pressure. The present study examined if the pressor response to l-cysteine is stereospecific and involves recruitment of hypothalamic vasopressinergic neurons and medullary noradrenergic A1 neurons. Intracisternally injected d-cysteine produced no cardiovascular changes, while l-cysteine produced hypertension and tachycardia in freely moving rats, indicating the stereospecific hemodynamic actions of l-cysteine via the brain. The double labeling immunohistochemistry combined with c-Fos detection as a marker of neuronal activation revealed significantly higher numbers of c-Fos-positive vasopressinergic neurons both in the supraoptic and paraventricular nuclei and tyrosine hydroxylase containing medullary A1 neurons, of l-cysteine-injected rats than those injected with d-cysteine as iso-osmotic control. The results indicate that the cardiovascular responses to intracisternal injection of l-cysteine in the conscious rat are stereospecific and include recruitment of hypothalamic vasopressinergic neurons both in the supraoptic and paraventricular nuclei, as well as of medullary A1 neurons. The findings may suggest a potential function of l-cysteine as an extracellular signal such as neuromodulators in central regulation of blood pressure.     

A combinatorial approach to the structure elucidation of a pyoverdine siderophore produced by a Pseudomonas putida isolate and the use of pyoverdine as a taxonomic marker for typing P. putida subspecies

The structure of a pyoverdine produced by Pseudomonas putida, W15Oct28, was elucidated by combining mass spectrometric methods and bioinformatics by the analysis of non-ribosomal peptide synthetase genes present in the newly sequenced genome. The only form of pyoverdine produced by P. putida W15Oct28 is characterized to contain α-ketoglutaric acid as acyl side chain, a dihydropyoverdine chromophore, and a 12 amino acid peptide chain. The peptide chain is unique among all pyoverdines produced by Pseudomonas subspecies strains. It was characterized as –l-Asp-l-Ala-d-AOHOrn-l-Thr-Gly-c[l-Thr(O-)-l-Hse-d-Hya-l-Ser-l-Orn-l-Hse-l-Ser-O-]. The chemical formula and the detected and calculated molecular weight of this pyoverdine are: C₆₅H₉₃N₁₇O₃₂, detected mass 1624.6404 Da, calculated mass 1624.6245. Additionally, pyoverdine structures from both literature reports and bioinformatics prediction of the genome sequenced P. putida strains are summarized allowing us to propose a scheme based on pyoverdines structures as tool for the phylogeny of P. putida. This study shows the strength of the combination of in silico analysis together with analytical data and literature mining in determining the structure of secondary metabolites such as peptidic siderophores.     

Probing the specificity of gamma-glutamylamine cyclotransferase: an enzyme involved in the metabolism of transglutaminase-catalyzed protein crosslinks

γ-Glutamylamine cyclotransferase (gGACT) catalyzes the intramolecular cyclization of a variety of l-γ-glutamylamines producing 5-oxo-l-proline and free amines. Its substrate specificity implicates it in the downstream metabolism of transglutaminase products, and is distinct from that of γ-glutamyl cyclotransferase which acts on l-γ-glutamyl amino acids. To elucidate the mechanism by which gGACT distinguishes between l-γ-glutamylamine and amino acid substrates, the specificity of the rabbit kidney enzyme for the amide region of substrates was probed through the kinetic analysis of a series of l-γ-glutamylamines. The isodipeptide N ^?-(l-γ-glutamyl)-l-lysine 1 was used as a reference. The kinetic constants of the l-γ-glutamyl derivative of n-butylamine 7, were nearly identical to those of 1. Introduction of a methyl or carboxylate group on the carbon adjacent to the side-chain amide nitrogen in l-γ-glutamylamine substrates resulted in a dramatic decrease in substrate properties for gGACT thus providing an explanation of why gGACT does not act on l-γ-glutamyl amino acids except for l-γ-glutamylglycine. Placement of substituents on carbons further removed from the side-chain amide nitrogen in l-γ-glutamylamines restored activity for gGACT, and l-γ-glutamylneohexylamine 19 had a higher specificity constant (k _cat /K _m) than 1. gGACT did not exhibit any stereospecificity in the amide region of l-γ-glutamylamine substrates. In addition, analogues (26–30) with heteroatom substitutions for the γ methylene position of the l-γ-glutamyl moiety were examined. Several thiocarbamoyl derivatives of l-cysteine (28–30) were excellent substrates for gGACT.     

Coupled bioconversion for preparation of N-acetyl-d-neuraminic acid using immobilized N-acetyl-d-glucosamine-2-epimerase and N-acetyl-d-neuraminic acid lyase

N-Acetyl-d-neuraminic acid (Neu5Ac) can be produced from N-acetyl-d-glucosamine (GlcNAc) and pyruvate by a chemoenzymatic process in which an alkaline-catalyzed epimerization transforms GlcNAc to N-acetyl-d-manosamine (ManNAc). ManNAc is then condensed biocatalytically with pyruvate in the presence of N-acetyl-d-neuraminic acid lyase (NAL) or by a two-step, fully enzymatic process involving bioconversions of GlcNAc to ManNAc and ManNAc to Neu5Ac using N-acetyl-d-glucosamine 2-epimerase (AGE) and NAL. There are some drawbacks to this technique, such as lengthy reaction time, and the low conversion rate when the soluble forms of the enzymes are used in the two-step enzymatic process. In this study, the Escherichia coli-expressed AGE and NAL in the supernatant were purified by FP-based affinity chromatography and then immobilized on Amberzyme oxirane resin. These two immobilized enzymes, with a specific activity of 78.18 U/g for AGE and 69.30 U/g for NAL, were coupled to convert GlcNAc to Neu5Ac directly in one reactor. The conversion rate of the two-step reactions from GlcNAc to Neu5Ac was ～73% within 24 h. Furthermore, the immobilized AGE and NAL could both be used up to five reaction cycles without loss of activity or significant decrease of the conversion rate.     

An l-glucitol oxidizing dehydrogenase from Bradyrhizobium japonicum USDA 110 for production of d-sorbose with enzymatic or electrochemical cofactor regeneration

A gene in Bradyrhizobium japonicum USDA 110, annotated as a ribitol dehydrogenase (RDH), had 87 % sequence identity (97 % positives) to the N-terminal 31 amino acids of an l-glucitol dehydrogenase from Stenotrophomonas maltophilia DSMZ 14322. The 729-bp long RDH gene coded for a protein consisting of 242 amino acids with a molecular mass of 26.1 kDa. The heterologously expressed protein not only exhibited the main enantio selective activity with d-glucitol oxidation to d-fructose but also converted l-glucitol to d-sorbose with enzymatic cofactor regeneration and a yield of 90 %. The temperature stability and the apparent K _m value for l-glucitol oxidation let the enzyme appear as a promising subject for further improvement by enzyme evolution. We propose to rename the enzyme from the annotated RDH gene (locus tag bll6662) from B. japonicum USDA as a d-sorbitol dehydrogenase (EC 1.1.1.14).     

The lipophilic vitamin C derivative, 6-o-palmitoylascorbate,protects human lymphocytes,preferentially over ascorbate,against X-ray-induced DNA damage,lipid peroxidation,and protein carbonylation

The aim of this study was to investigate protective effects of the lipophilic vitamin C derivative, 6-o-palmitoylascorbate (PlmtVC), against X-ray radiation-induced damages including cell death, DNA double-strand breaks (DSBs), lipid peroxidation, and protein carbonylation in human lymphocytes HEV0082, and the stability of PlmtVC under cell-cultured or cell-free condition. Irradiation with X-ray (1.5 Gy) diminished the cell viability and induced apoptosis, both of which were protected by pre-irradiational administration with PlmtVC. Gamma-H2A.X foci as a hallmark of DSBs were markedly enhanced in the irradiated cells. PlmtVC prevented X-ray-induced DSBs more appreciably than l-ascorbic acid (l-AA). Intracellular ROS production, lipid peroxidation, and protein carbonylation in HEV0082 cells were increased by X-ray at 1.5 Gy, all of which were significantly repressed by PlmtVC. PlmtVC also elevated endogenous reduced glutathione (GSH) in HEV0082 cells, and prevented X-ray-induced GSH depletion that are more appreciably over l-AA. Thus, PlmtVC prevents X-ray-induced cell death through its antioxidative activity. Stability tests showed that after being kept under physiological conditions (pH 7.4, 37 °C) for 14 days, vitamin C residual rates in PlmtVC solutions (62.2–82.0 %) were significantly higher than those in l-AA solutions (20.5–28.7 %). When PlmtVC or l-AA was added to HEV0082 lymphocytes, intracellular vitamin C in l-AA-treated cells was not detectable after 24 h, whereas PlmtVC-treated cells could keep a high level of intracellular vitamin C, suggesting an excellent stability of PlmtVC. Thus, X-ray-induced diverse harmful effects could be prevented by PlmtVC, which was suggested to ensue intrinsically from the persistent enrichment of intracellular vitamin C, resulting in relief to X-ray-caused oxidative stress.     

Alanine-scanning mutation approach for classification of the roles of conserved residues in the activity and substrate affinity of l-carnitine dehydrogenase

l-Carnitine dehydrogenase (CDH) is as an excellent tool for l-carnitine (l-Car) estimation. To date, four CDHs have been identified, that share 45 % homology of their proteins. Here 42 conserved residues of CDH from Xanthomonas translucens (Xt-CDH) were substituted successively with alanine. The resultant mutants were analyzed for catalytic activity. Active mutants were evaluated for their influence on l-Car affinity. Twenty-three mutants with reduced affinity toward l-Car were subjected to detailed kinetic analysis. Analytical data implied that all mutants had increased K _m values. The mutants of R193A, E196A, W199A, R200A, F249A, and F253A that produced the greatest l-Car affinity disruption (K _m > 200-folds of Xt-CDH) clustered near the putative active site. This information can provide a solid basis for the rational design of mutagenic investigation to improve CDHs.     

Optimization of Biotransformation of l-Tyrosine to l-DOPA by Yarrowia lipolytica-NCIM 3472 Using Response Surface Methodology

l-DOPA (3,4-dihydroxyphenyl-l-alanine) is the most widely used drug for treatment of Parkinson’s disease. In this study Yarrowia lipolytica-NCIM 3472 biomass was used for transformation of l-tyrosine to l-DOPA. The process parameters were optimized using response surface methodology (RSM). The optimum values of the tested variables for the production of l-DOPA were: pH 7.31, temperature 42.9 °C, 2.31 g l^?1 cell mass and 1.488 g l^?1 l-tyrosine. The highest yield obtained with these optimum parameters along with recycling of the cells was 4.091 g l^?1. This optimization of process parameters using RSM resulted in 4.609-fold increase in the l-DOPA production. The statistical analysis showed that the model was significant. Also coefficient of determination (R²) was 0.9758, indicating a good agreement between the experimental and predicted values of l-DOPA production. The highest tyrosinase activity observed was 7,028 U mg^?1 tyrosine. l-DOPA production was confirmed by HPTLC and HPLC analysis. Thus, RSM approach effectively enhanced the potential of Y. lipolytica-NCIM 3472 as an alternative source to produce l-DOPA.