Circulating N-lactoyl-amino acids and N-formyl-methionine reflect mitochondrial dysfunction and predict mortality in septic shock.

INTRODUCTION: Sepsis is a highly morbid condition characterized by multi-organ dysfunction resulting from dysregulated inflammation in response to acute infection. Mitochondrial dysfunction may contribute to sepsis pathogenesis, but quantifying mitochondrial dysfunction remains challenging. OBJECTIVE: To assess the extent to which circulating markers of mitochondrial dysfunction are increased in septic shock, and their relationship to severity and mortality. METHODS: We performed both full-scan and targeted (known markers of genetic mitochondrial disease) metabolomics on plasma to determine markers of mitochondrial dysfunction which distinguish subjects with septic shock (n = 42) from cardiogenic shock without infection (n = 19), bacteremia without sepsis (n = 18), and ambulatory controls (n = 19) - the latter three being conditions in which mitochondrial function, proxied by peripheral oxygen consumption, is presumed intact. RESULTS: Nine metabolites were significantly increased in septic shock compared to all three comparator groups. This list includes N-formyl-L-methionine (f-Met), a marker of dysregulated mitochondrial protein translation, and N-lactoyl-phenylalanine (lac-Phe), representative of the N-lactoyl-amino acids (lac-AAs), which are elevated in plasma of patients with monogenic mitochondrial disease. Compared to lactate, the clinical biomarker used to define septic shock, there was greater separation between survivors and non-survivors of septic shock for both f-Met and the lac-AAs measured within 24 h of ICU admission. Additionally, tryptophan was the one metabolite significantly decreased in septic shock compared to all other groups, while its breakdown product kynurenate was one of the 9 significantly increased. CONCLUSION: Future studies which validate the measurement of lac-AAs and f-Met in conjunction with lactate could define a sepsis subtype characterized by mitochondrial dysfunction.

The major role of Listeria monocytogenes folic acid metabolism during infection is the generation of N-formylmethionine.

IMPORTANCE: Folic acid is an essential vitamin for bacteria, plants, and animals. The lack of folic acid leads to various consequences such as a shortage of amino acids and nucleotides that are fundamental building blocks for life. Though antifolate drugs are widely used for antimicrobial treatments, the underlying mechanism of bacterial folate deficiency during infection is unclear. This study compares the requirements of different folic acid end-products during the infection of Listeria monocytogenes, a facultative intracellular pathogen of animals and humans. The results reveal the critical importance of N-formylmethionine, the amino acid used by bacteria to initiate protein synthesis. This work extends the current understanding of folic acid metabolism in pathogens and potentially provides new insights into antifolate drug development in the future.

Mitochondrial N-formyl methionine peptides contribute to exaggerated neutrophil activation in patients with COVID-19.

Neutrophil dysregulation is well established in COVID-19. However, factors contributing to neutrophil activation in COVID-19 are not clear. We assessed if N-formyl methionine (fMet) contributes to neutrophil activation in COVID-19. Elevated levels of calprotectin, neutrophil extracellular traps (NETs) and fMet were observed in COVID-19 patients (n = 68), particularly in critically ill patients, as compared to HC (n = 19, p < 0.0001). Of note, the levels of NETs were higher in ICU patients with COVID-19 than in ICU patients without COVID-19 (p < 0.05), suggesting a prominent contribution of NETs in COVID-19. Additionally, plasma from COVID-19 patients with mild and moderate/severe symptoms induced in vitro neutrophil activation through fMet/FPR1 (formyl peptide receptor-1) dependent mechanisms (p < 0.0001). fMet levels correlated with calprotectin levels validating fMet-mediated neutrophil activation in COVID-19 patients (r = 0.60, p = 0.0007). Our data indicate that fMet is an important factor contributing to neutrophil activation in COVID-19 disease and may represent a potential target for therapeutic intervention.

Deformylation of nascent peptide chains on the ribosome.

Processing of newly synthesized polypeptides is essential for protein homeostasis and cell viability. In bacteria and eukaryotic organelles, all proteins are synthesized with formylmethionine at their N-terminus. As the nascent peptide emerges from the ribosome during translation, the formyl group is removed by peptide deformylase (PDF), an enzyme that belongs to the family of ribosome-associated protein biogenesis factors (RPBs). Because PDF is essential in bacteria but not in humans (except for the PDF homolog acting in mitochondria), the bacterial enzyme is a promising antimicrobial drug target. While much of the mechanistic work on PDF was carried out using model peptides in solution, understanding the mechanism of PDF in cells and developing effective PDF inhibitors requires experiments with its native cellular substrates, i.e., ribosome-nascent chain complexes. Here, we describe protocols to purify PDF from Escherichia coli and to test its deformylation activity on the ribosome in multiple-turnover and single-round kinetic regimes as well as in binding assays. These protocols can be used to test PDF inhibitors, to study the peptide specificity of PDF and its interplay with other RPBs, as well as to compare the activity and specificity of bacterial and mitochondrial PDFs.

Detection of Nα-terminally formylated native proteins by a pan-N-formyl methionine-specific antibody.

N-formyl methionine (fMet)-containing proteins are produced in bacteria, eukaryotic organelles mitochondria and plastids, and even in cytosol. However, Nα-terminally formylated proteins have been poorly characterized because of the lack of appropriate tools to detect fMet independently of downstream proximal sequences. Using a fMet-Gly-Ser-Gly-Cys peptide as an antigen, we generated a pan-fMet-specific rabbit polyclonal antibody called anti-fMet. The raised anti-fMet recognized universally and sequence context-independently Nt-formylated proteins in bacterial, yeast, and human cells as determined by a peptide spot array, dot blotting, and immunoblotting. We anticipate that the anti-fMet antibody will be broadly used to enable an understanding of the poorly explored functions and mechanisms of Nt-formylated proteins in various organisms.

Circulating N-formylmethionine and metabolic shift in critical illness: a multicohort metabolomics study.

BACKGROUND: Cell stress promotes degradation of mitochondria which release danger-associated molecular patterns that are catabolized to N-formylmethionine. We hypothesized that in critically ill adults, the response to N-formylmethionine is associated with increases in metabolomic shift-related metabolites and increases in 28-day mortality. METHODS: We performed metabolomics analyses on plasma from the 428-subject Correction of Vitamin D Deficiency in Critically Ill Patients trial (VITdAL-ICU) cohort and the 90-subject Brigham and Women's Hospital Registry of Critical Illness (RoCI) cohort. In the VITdAL-ICU cohort, we analyzed 983 metabolites at Intensive Care Unit (ICU) admission, day 3, and 7. In the RoCI cohort, we analyzed 411 metabolites at ICU admission. The association between N-formylmethionine and mortality was determined by adjusted logistic regression. The relationship between individual metabolites and N-formylmethionine abundance was assessed with false discovery rate correction via linear regression, linear mixed-effects, and Gaussian graphical models. RESULTS: Patients with the top quartile of N-formylmethionine abundance at ICU admission had a significantly higher adjusted odds of 28-day mortality in the VITdAL-ICU (OR, 2.4; 95%CI 1.5-4.0; P = 0.001) and RoCI cohorts (OR, 5.1; 95%CI 1.4-18.7; P = 0.015). Adjusted linear regression shows that with increases in N-formylmethionine abundance at ICU admission, 55 metabolites have significant differences common to both the VITdAL-ICU and RoCI cohorts. With increased N-formylmethionine abundance, both cohorts had elevations in individual short-chain acylcarnitine, branched chain amino acid, kynurenine pathway, and pentose phosphate pathway metabolites. CONCLUSIONS: The results indicate that circulating N-formylmethionine promotes a metabolic shift with heightened mortality that involves incomplete mitochondrial fatty acid oxidation, increased branched chain amino acid metabolism, and activation of the pentose phosphate pathway.

Mitochondrial DNA variants modulate N-formylmethionine, proteostasis and risk of late-onset human diseases.

Mitochondrial DNA (mtDNA) variants influence the risk of late-onset human diseases, but the reasons for this are poorly understood. Undertaking a hypothesis-free analysis of 5,689 blood-derived biomarkers with mtDNA variants in 16,220 healthy donors, here we show that variants defining mtDNA haplogroups Uk and H4 modulate the level of circulating N-formylmethionine (fMet), which initiates mitochondrial protein translation. In human cytoplasmic hybrid (cybrid) lines, fMet modulated both mitochondrial and cytosolic proteins on multiple levels, through transcription, post-translational modification and proteolysis by an N-degron pathway, abolishing known differences between mtDNA haplogroups. In a further 11,966 individuals, fMet levels contributed to all-cause mortality and the disease risk of several common cardiovascular disorders. Together, these findings indicate that fMet plays a key role in common age-related disease through pleiotropic effects on cell proteostasis.

Pseudouridine and N-formylmethionine associate with left ventricular mass index: Metabolome-wide association analysis of cardiac remodeling.

BACKGROUND: Heart failure (HF) is the fastest growing form of cardiovascular disease both nationally and globally, underlining a need to phenotype subclinical HF intermediaries to improve primary prevention. OBJECTIVES: We aimed to identify novel metabolite associations with left ventricular (LV) remodeling, one upstream HF intermediary, among a community-based cohort of individuals. METHODS: We examined 1052 Bogalusa Heart Study participants (34.98% African American, 57.41% female, aged 33.6-57.5 years). Measures of LV mass and relative wall thickness (RWT) were obtained using two-dimensional-guided echocardiographic measurements via validated eqs. LV mass was indexed to height2.7 to calculate left ventricular mass index (LVMI). Untargeted metabolomic analysis of fasting serum samples was conducted. In combined and ethnicity-stratified analyses, multivariable linear and multinomial logistic regression models tested the associations of metabolites with the continuous LVMI and RWT and categorical LV geometry phenotypes, respectively, after adjusting for demographic and traditional cardiovascular disease risk factors. RESULTS: Pseudouridine (B = 1.38; p = 3.20 × 10-5) and N-formylmethionine (B = 1.65; 3.30 × 10-6) were significantly associated with LVMI in the overall sample as well significant in Caucasians, with consistent effect direction and nominal significance (p < .05) in African Americans. Upon exclusion of individuals with self-report myocardial infarction or congestive HF, we similarly observed a 1.33 g/m2.7 and 1.52 g/m2.7 higher LVMI for each standard deviation increase in pseudouridine and N-formylmethionine, respectively. No significant associations were observed for metabolites with RWT or categorical LV remodeling outcomes. CONCLUSIONS: The current analysis identified novel associations of pseudouridine and N-formylmethionine with LVMI, suggesting that mitochondrial-derived metabolites may serve as early biomarkers for LV remodeling and subclinical HF.

Reiterative Synthesis by the Ribosome and Recognition of the N-Terminal Formyl Group by Biosynthetic Machinery Contribute to Evolutionary Conservation of the Length of Antibiotic Microcin C Peptide Precursor.

Microcin C (McC) is a peptide adenylate antibiotic produced by Escherichiacoli cells bearing a plasmid-borne mcc gene cluster. Most MccA precursors, encoded by validated mcc operons from diverse bacteria, are 7 amino acids long, but the significance of this precursor length conservation has remained unclear. Here, we created derivatives of E. colimcc operons encoding longer precursors and studied their synthesis and bioactivities. We found that increasing the precursor length to 11 amino acids and beyond strongly decreased antibiotic production. We found this decrease to depend on several parameters. First, reiterative synthesis of the MccA peptide by the ribosome was decreased at longer mccA open reading frames, leading to less efficient competition with other messenger RNAs. Second, the presence of a formyl group at the N-terminal methionine of the heptameric peptide had a strong stimulatory effect on adenylation by the MccB enzyme. No such formyl group stimulation was observed for longer peptides. Finally, the presence of the N-terminal formyl on the heptapeptide adenylate stimulated bioactivity, most likely at the uptake stage. Together, these factors should contribute to optimal activity of McC-like compounds as 7-amino-acid peptide moieties and suggest convergent evolution of several steps of the antibiotic biosynthesis pathway and their adjustment to sensitive cell uptake machinery to create a potent drug.IMPORTANCEEscherichia coli microcin C (McC) is a representative member of peptide-nucleotide antibiotics produced by diverse microorganisms. The vast majority of biosynthetic gene clusters responsible for McC-like compound production encode 7-amino-acid-long precursor peptides, which are C-terminally modified by dedicated biosynthetic enzymes with a nucleotide moiety to produce a bioactive compound. In contrast, the sequences of McC-like compound precursor peptides are not conserved. Here, we studied the consequences of E. coli McC precursor peptide length increase on antibiotic production and activity. We show that increasing the precursor peptide length strongly decreases McC production by affecting multiple biosynthetic steps, suggesting that the McC biosynthesis system has evolved under significant functional constraints to maintain the precursor peptide length.

Formyl-methionine as an N-degron of a eukaryotic N-end rule pathway.

In bacteria, nascent proteins bear the pretranslationally generated N-terminal (Nt) formyl-methionine (fMet) residue. Nt-fMet of bacterial proteins is a degradation signal, termed fMet/N-degron. By contrast, proteins synthesized by cytosolic ribosomes of eukaryotes were presumed to bear unformylated Nt-Met. Here we found that the yeast formyltransferase Fmt1, although imported into mitochondria, could also produce Nt-formylated proteins in the cytosol. Nt-formylated proteins were strongly up-regulated in stationary phase or upon starvation for specific amino acids. This up-regulation strictly required the Gcn2 kinase, which phosphorylates Fmt1 and mediates its retention in the cytosol. We also found that the Nt-fMet residues of Nt-formylated proteins act as fMet/N-degrons and identified the Psh1 ubiquitin ligase as the recognition component of the eukaryotic fMet/N-end rule pathway, which destroys Nt-formylated proteins.

Mice deficient in the Shmt2 gene have mitochondrial respiration defects and are embryonic lethal.

Accumulation of somatic mutations in mitochondrial DNA (mtDNA) has been proposed to be responsible for human aging and age-associated mitochondrial respiration defects. However, our previous findings suggested an alternative hypothesis of human aging-that epigenetic changes but not mutations regulate age-associated mitochondrial respiration defects, and that epigenetic downregulation of nuclear-coded genes responsible for mitochondrial translation [e.g., glycine C-acetyltransferase (GCAT), serine hydroxymethyltransferase 2 (SHMT2)] is related to age-associated respiration defects. To examine our hypothesis, here we generated mice deficient in Gcat or Shmt2 and investigated whether they have respiration defects and premature aging phenotypes. Gcat-deficient mice showed no macroscopic abnormalities including premature aging phenotypes for up to 9 months after birth. In contrast, Shmt2-deficient mice showed embryonic lethality after 13.5 days post coitum (dpc), and fibroblasts obtained from 12.5-dpc Shmt2-deficient embryos had respiration defects and retardation of cell growth. Because Shmt2 substantially controls production of N-formylmethionine-tRNA (fMet-tRNA) in mitochondria, its suppression would reduce mitochondrial translation, resulting in expression of the respiration defects in fibroblasts from Shmt2-deficient embryos. These findings support our hypothesis that age-associated respiration defects in fibroblasts of elderly humans are caused not by mtDNA mutations but by epigenetic regulation of nuclear genes including SHMT2.

Poly-MVA attenuates 7,12- dimethylbenz[a]anthracene initiated and croton oil promoted skin papilloma formation on mice skin.

OBJECTIVE: Chemopreventive agents which exhibit activities such as anti-inflammation, inhibition of carcinogen induced mutagenesis and scavenging of free radical might play a decisive role in the inhibition of chemical carcinogenesis either at the initiation or promotion stage. Many synthesized palladium (Pd) complexes tested experimentally for antitumor activity are found effective. Poly-MVA is a liquid blend preparation containing B complex vitamins, ruthenium with Pd complexed with alpha lipoic acid as the major ingredients. The antitumor effect of Poly-MVA was evaluated against 7,12-dimethylbenz[a] anthracene-initiated croton oil-promoted papilloma formation on mice skin. Skin tumor was initiated with a single application of 390 nmol of DMBA in 20 µl acetone. The effect of Poly-MVA against croton oil- induced inflammation and lipid peroxidation on the mice skin was also evaluated. Topical application of Poly-MVA (100 µl, twice weekly for 18 weeks) 30 minutes prior to each croton oil application, significantly decreased the tumor incidence (11%) and the average number of tumor per animals. Application of Poly-MVA (100 µl) before croton oil significantly (p &#60; 0.05) protected the mouse skin from inflammation (36%) and lipid peroxidation (14%) when compared to the croton oil alone treated group. Experimental results indicate that Poly-MVA attenuate the tumor promoting effects of croton oil and the effect may probably be due to its anti-inflammatory and antioxidant activity.

Lack of formylated methionyl-tRNA has pleiotropic effects on Bacillus subtilis.

Bacteria initiate translation using a modified amino acid, N-formylmethionine (fMet), adapted specifically for this function. Most proteins are processed co-translationally by peptide deformylase (PDF) to remove this modification. Although PDF activity is essential in WT cells and is the target of the antibiotic actinonin, bypass mutations in the fmt gene that eliminate the formylation of Met-tRNAMet render PDF dispensable. The extent to which the emergence of fmt bypass mutations might compromise the therapeutic utility of actinonin is determined, in part, by the effects of these bypass mutations on fitness. Here, we characterize the phenotypic consequences of an fmt null mutation in the model organism Bacillus subtilis. An fmt null mutant is defective for several post-exponential phase adaptive programmes including antibiotic resistance, biofilm formation, swarming and swimming motility and sporulation. In addition, a survey of well-characterized stress responses reveals an increased sensitivity to metal ion excess and oxidative stress. These diverse phenotypes presumably reflect altered synthesis or stability of key proteins involved in these processes.

Peptide length and folding state govern the capacity of staphylococcal ß-type phenol-soluble modulins to activate human formyl-peptide receptors 1 or 2.

Most staphylococci produce short α-type PSMs and about twice as long ß-type PSMs that are potent leukocyte attractants and toxins. PSMs are usually secreted with the N-terminal formyl group but are only weak agonists for the leukocyte FPR1. Instead, the FPR1-related FPR2 senses PSMs efficiently and is crucial for leukocyte recruitment in infection. Which structural features distinguish FPR1 from FPR2 ligands has remained elusive. To analyze which peptide properties may govern the capacities of ß-type PSMs to activate FPRs, full-length and truncated variants of such peptides from Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus lugdunensis were synthesized. FPR2 activation was observed even for short N- or C-terminal ß-type PSM variants once they were longer than 18 aa, and this activity increased with length. In contrast, the shortest tested peptides were potent FPR1 agonists, and this property declined with increasing peptide length. Whereas full-length ß-type PSMs formed α-helices and exhibited no FPR1-specific activity, the truncated peptides had less-stable secondary structures, were weak agonists for FPR1, and required N-terminal formyl-methionine residues to be FPR2 agonists. Together, these data suggest that FPR1 and FPR2 have opposed ligand preferences. Short, flexible PSM structures may favor FPR1 but not FPR2 activation, whereas longer peptides with α-helical, amphipathic properties are strong FPR2 but only weak FPR1 agonists. These findings should help to unravel the ligand specificities of 2 critical human PRRs, and they may be important for new, anti-infective and anti-inflammatory strategies.

Role of N-terminal protein formylation in central metabolic processes in Staphylococcus aureus.

BACKGROUND: Bacterial protein biosynthesis usually depends on a formylated methionyl start tRNA but Staphylococcus aureus is viable in the absence of Fmt, the tRNAMet formyl transferase. fmt mutants exhibit reduced growth rates indicating that the function of certain proteins depends on formylated N-termini but it has remained unclear, which cellular processes are abrogated by the lack of formylation. RESULTS: In order to elucidate how global metabolic processes are affected by the absence of formylated proteins the exometabolome of an S. aureus fmt mutant was compared with that of the parental strain and the transcription of corresponding enzymes was analyzed to identify possible regulatory changes. The mutant consumed glucose and other carbon sources slower than the wild type. While the turnover of several metabolites remained unaltered fmt inactivation led to increases pyruvate release and, concomitantly, reduced pyruvate dehydrogenase activity. In parallel, the release of the pyruvate-derived metabolites lactate, acetoin, and alanine was reduced. The anaerobic degradation of arginine was also reduced in the fmt mutant compared to the wild-type strain. Moreover, the lack of formylated proteins caused increased susceptibility to the antibiotics trimethoprim and sulamethoxazole suggesting that folic acid-dependant pathways were perturbed in the mutant. CONCLUSIONS: These data indicate that formylated proteins are crucial for specific bacterial metabolic processes and they may help to understand why it has remained important during bacterial evolution to initiate protein biosynthesis with a formylated tRNAMet.

Predict prokaryotic proteins through detecting N-formylmethionine residues in protein sequences using support vector machine.

Identifying prokaryotes in silico is commonly based on DNA sequences. In experiments where DNA sequences may not be immediately available, we need to have a different approach to detect prokaryotes based on RNA or protein sequences. N-formylmethionine (fMet) is known as a typical characteristic of prokaryotes. A web tool has been implemented here for predicting prokaryotes through detecting the N-formylmethionine residues in protein sequences. The predictor is constructed using support vector machine. An online predictor has been implemented using Python. The implemented predictor is able to achieve the total prediction accuracy 80% with the specificity 80% and the sensitivity 81%.

A peptide deformylase-ribosome complex reveals mechanism of nascent chain processing.

Messenger-RNA-directed protein synthesis is accomplished by the ribosome. In eubacteria, this complex process is initiated by a specialized transfer RNA charged with formylmethionine (tRNA(fMet)). The amino-terminal formylated methionine of all bacterial nascent polypeptides blocks the reactive amino group to prevent unfavourable side-reactions and to enhance the efficiency of translation initiation. The first enzymatic factor that processes nascent chains is peptide deformylase (PDF); it removes this formyl group as polypeptides emerge from the ribosomal tunnel and before the newly synthesized proteins can adopt their native fold, which may bury the N terminus. Next, the N-terminal methionine is excised by methionine aminopeptidase. Bacterial PDFs are metalloproteases sharing a conserved N-terminal catalytic domain. All Gram-negative bacteria, including Escherichia coli, possess class-1 PDFs characterized by a carboxy-terminal alpha-helical extension. Studies focusing on PDF as a target for antibacterial drugs have not revealed the mechanism of its co-translational mode of action despite indications in early work that it co-purifies with ribosomes. Here we provide biochemical evidence that E. coli PDF interacts directly with the ribosome via its C-terminal extension. Crystallographic analysis of the complex between the ribosome-interacting helix of PDF and the ribosome at 3.7 A resolution reveals that the enzyme orients its active site towards the ribosomal tunnel exit for efficient co-translational processing of emerging nascent chains. Furthermore, we have found that the interaction of PDF with the ribosome enhances cell viability. These results provide the structural basis for understanding the coupling between protein synthesis and enzymatic processing of nascent chains, and offer insights into the interplay of PDF with the ribosome-associated chaperone trigger factor.

Kinetically competent intermediates in the translocation step of protein synthesis.

Translocation requires large-scale movements of ribosome-bound tRNAs. Using tRNAs that are proflavin labeled and single-turnover rapid kinetics assays, we identify one or possibly two kinetically competent intermediates in translocation. EF-G.GTP binding to the pretranslocation (PRE) complex and GTP hydrolysis are rapidly followed by formation of the securely identified intermediate complex (INT), which is more slowly converted to the posttranslocation (POST) complex. Peptidyl tRNA within the INT complex occupies a hybrid site, which has a puromycin reactivity intermediate between those of the PRE and POST complexes. Thiostrepton and viomycin inhibit INT formation, whereas spectinomycin selectively inhibits INT disappearance. The effects of other translocation modulators suggest that EF-G-dependent GTP hydrolysis is more important for INT complex formation than for INT complex conversion to POST complex and that subtle changes in tRNA structure influence coupling of tRNA movement to EF-G.GTP-induced conformational changes.

Molecular cloning and biochemical characterization of L-N-carbamoylase from Sinorhizobium meliloti CECT4114.

An N-carbamoyl-L-amino acid amidohydrolase (L-N-carbamoylase) from Sinorhizobium meliloti CECT 4114 was cloned and expressed in Escherichia coli. The recombinant enzyme catalyzed the hydrolysis of N-carbamoyl alpha-amino acid to the corresponding free amino acid, and its purification has shown it to be strictly L-specific. The enzyme showed broad substrate specificity, and it is the first L-N-carbamoylase that hydrolyses N-carbamoyl-L-tryptophan as well as N-carbamoyl L-amino acids with aliphatic substituents. The apparent Km values for N-carbamoyl-L-methionine and tryptophan were very similar (0.65 +/- 0.09 and 0.69 +/- 0.08 mM, respectively), although the rate constant was clearly higher for the L-methionine precursor (14.46 +/- 0.30 s(-1)) than the L-tryptophan one (0.15 +/- 0.01 s(-1)). The enzyme also hydrolyzed N-formyl-L-methionine (kcat/Km = 7.10 +/- 2.52 s(-1) x mM(-1)) and N-acetyl-L-methionine (kcat/Km = 12.16 +/- 1.93 s(-1) x mM(-1)), but the rate of hydrolysis was lower than for N-carbamoyl-L-methionine (kcat/Km = 21.09 +/- 2.85). This is the first L-N-carbamoylase involved in the 'hydantoinase process' that has hydrolyzed N-carbamoyl-L-cysteine, though less efficiently than N-carbamoyl-L-methionine. The enzyme did not hydrolyze ureidosuccinic acid or 3-ureidopropionic acid. The native form of the enzyme was a homodimer with a molecular mass of 90 kDa. The optimum conditions for the enzyme were 60 degrees C and pH 8.0. Enzyme activity required the presence of divalent metal ions such as Ni2+, Mn2+, Co2+ and Fe2+, and five amino acids putatively involved in the metal binding were found in the amino acid sequence.